Anti-cmet antagonists

ABSTRACT

The invention provides therapeutic anti-c-met antibodies, and compositions comprising and methods of using these antibodies.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/008,836, filed Jan. 18, 2011 which is a divisional application ofU.S. patent application Ser. No. 11/537,760, filed Oct. 2, 2006, nowU.S. Pat. No. 7,892,550, which is a continuation of U.S. patentapplication Ser. No. 11/196,917, filed on Aug. 4, 2005, now U.S. Pat.No. 7,476,724, which application claims priority to provisionalapplication No. 60/598,991, filed Aug. 5, 2004, the contents of whichare incorporated herein in their entirety by reference.

TECHNICAL FIELD

The present invention relates generally to the fields of molecularbiology and growth factor regulation. More specifically, the inventionconcerns modulators of the HGF/c-met signaling pathway, and uses of saidmodulators.

BACKGROUND

HGF is a mesenchyme-derived pleiotrophic factor with mitogenic,motogenic and morphogenic activities on a number of different celltypes. HGF effects are mediated through a specific tyrosine kinase,c-met, and aberrant HGF and c-met expression are frequently observed ina variety of tumors. See, e.g., Maulik et al., Cytokine & Growth FactorReviews (2002), 13:41-59; Danilkovitch-Miagkova & Zbar, J. Clin. Invest.(2002), 109(7):863-867. Regulation of the HGF/c-Met signaling pathway isimplicated in tumor progression and metastasis. See, e.g., Trusolino &Comoglio, Nature Rev. (2002), 2:289-300).

HGF binds the extracellular domain of the Met receptor tyrosine kinase(RTK) and regulates diverse biological processes such as cellscattering, proliferation, and survival. HGF-Met signaling is essentialfor normal embryonic development especially in migration of muscleprogenitor cells and development of the liver and nervous system (Bladtet al., 1995; Hamanoue et al., 1996; Maim et al., 1996; Schmidt et al.,1995; Uehara et al., 1995). Developmental phenotypes of Met and HGFknockout mice are very similar suggesting that HGF is the cognate ligandfor the Met receptor (Schmidt et al., 1995; Uehara et al., 1995).HGF-Met also plays a role in liver regeneration, angiogenesis, and woundhealing (Bussolino et al., 1992; Matsumoto and Nakamura, 1993; Nusrat etal., 1994). The precursor Met receptor undergoes proteolytic cleavageinto an extracellular subunit and membrane spanning subunit linked bydisulfide bonds (Tempest et al., 1988). The subunit contains thecytoplasmic kinase domain and harbors a multi-substrate docking site atthe C-terminus where adapter proteins bind and initiate signaling(Bardelli et al., 1997; Nguyen et al., 1997; Pelicci et al., 1995;Ponzetto et al., 1994; Weidner et al., 1996). Upon HGF binding,activation of Met leads to tyrosine phosphorylation and downstreamsignaling through Gab1 and Grb2/Sos mediated PI3-kinase and Ras/MAPKactivation respectively, which drives cell motility and proliferation(Furge et al., 2000; Hartmann et al., 1994; Ponzetto et al., 1996; Royaland Park, 1995).

Met was shown to be transforming in a carcinogen-treated osteosarcomacell line (Cooper et al., 1984; Park et al., 1986). Met overexpressionor gene-amplification has been observed in a variety of human cancers.For example, Met protein is overexpressed at least 5-fold in colorectalcancers and reported to be gene-amplified in liver metastasis (Di Renzoet al., 1995; Liu et al., 1992). Met protein is also reported to beoverexpressed in oral squamous cell carcinoma, hepatocellular carcinoma,renal cell carcinoma, breast carcinoma, and lung carcinoma (Jin et al.,1997; Morello et al., 2001; Natali et al., 1996; Olivero et al., 1996;Suzuki et al., 1994). In addition, overexpression of mRNA has beenobserved in hepatocellular carcinoma, gastric carcinoma, and colorectalcarcinoma (Boix et al., 1994; Kuniyasu et al., 1993; Liu et al., 1992).

A number of mutations in the kinase domain of Met have been found inrenal papillary carcinoma which leads to constitutive receptoractivation (Olivero et al., 1999; Schmidt et al., 1997; Schmidt et al.,1999). These activating mutations confer constitutive Met tyrosinephosphorylation and result in MAPK activation, focus formation, andtumorigenesis (Jeffers et al., 1997). In addition, these mutationsenhance cell motility and invasion (Giordano et al., 2000; Lorenzato etal., 2002). HGF-dependent Met activation in transformed cells mediatesincreased motility, scattering, and migration which eventually leads toinvasive tumor growth and metastasis (Jeffers et al., 1996; Meiners etal., 1998).

Met has been shown to interact with other proteins that drive receptoractivation, transformation, and invasion. In neoplastic cells, Met isreported to interact with 6 4 integrin, a receptor for extracellularmatrix (ECM) components such as laminins, to promote HGF-dependentinvasive growth (Trusolino et al., 2001). In addition, the extracellulardomain of Met has been shown to interact with a member of the semaphorinfamily, plexin B1, and to enhance invasive growth (Giordano et al.,2002). Furthermore, CD44v6, which has been implicated in tumorigenesisand metastasis, is also reported to form a complex with Met and HGF andresult in Met receptor activation (Orian-Rousseau et al., 2002).

Met is a member of the subfamily of RTKs which include Ron and Sea(Maulik et al., 2002). Prediction of the extracellular domain structureof Met suggests shared homology with the semaphorins and plexins. TheN-terminus of Met contains a Sema domain of approximately 500 aminoacids that is conserved in all semaphorins and plexins. The semaphorinsand plexins belong to a large family of secreted and membrane-boundproteins first described for their role in neural development (VanVactor and Lorenz, 1999). However, more recently semaphorinoverexpression has been correlated with tumor invasion and metastasis. Acysteine-rich PSI domain (also referred to as a Met Related Sequencedomain) found in plexins, semaphorins, and integrins lies adjacent tothe Sema domain followed by four IPT repeats that areimmunoglobulin-like regions found in plexins and transcription factors.A recent study suggests that the Met Sema domain is sufficient for HGFand heparin binding (Gherardi et al., 2003). Furthermore, Kong-Beltranet al. (Cancer Cell (2004), 6:61-73) have reported that the Sema domainof Met is necessary for receptor dimerization and activation.

Numerous molecules targeted at the HGF/c-met pathway have been reported.These molecules include antibodies such as those described in U.S. Pat.No. 5,686,292. A portion of the extracellular domain of c-met has alsobeen shown to be capable of antagonistic effects against the HGF/c-metpathway. In view of the important role that this pathway plays in theetiology of various pathological conditions, however, it is clear thatthere continues to be a need for agents that have clinical attributesthat are optimal for development as therapeutic agents. The inventiondescribed herein meets this need and provides other benefits.

All references cited herein, including patent applications andpublications, are incorporated by reference in their entirety.

DISCLOSURE OF THE INVENTION

The invention is in part based on the identification of a variety ofantagonists of the HGF/c-met biological pathway, which is abiological/cellular process that presents as an important andadvantageous therapeutic target. The invention provides compositions andmethods based on interfering with HGF/c-met activation, including butnot limited to interfering with HGF binding to the extracellular portionof c-met and receptor multimerization. Antagonists of the invention, asdescribed herein, provide important therapeutic and diagnostic agentsfor use in targeting pathological conditions associated with abnormal orunwanted signaling of the HGF/c-met pathway. Accordingly, the inventionprovides methods, compositions, kits and articles of manufacture relatedto modulating the HGF/c-met pathway, including modulation of c-metligand binding, c-met dimerization, activation, and otherbiological/physiological activities associated with HGF/c-met signaling.

In one aspect, the invention provides anti-HGF/c-met therapeutic agentssuitable for therapeutic use and capable of effecting varying degrees ofdisruption of the HGF/c-met signaling pathway. For example, in oneembodiment, the invention provides a humanized anti-c-met antibodywherein the monovalent affinity of the antibody to human c-met (e.g.,affinity of the antibody as a Fab fragment to human c-met) issubstantially the same as the monovalent affinity of a murine antibody(e.g., affinity of the murine antibody as a Fab fragment to human c-met)comprising, consisting or consisting essentially of a light chain andheavy chain variable domain sequence as depicted in FIG. 7 (SEQ ID NO:9and 10). In another embodiment, the invention provides a humanizedanti-c-met antibody wherein the monovalent affinity of the antibody tohuman c-met (e.g., affinity of the antibody as a Fab fragment to humanc-met) is lower, for example at least 3, 5, 7 or 10-fold lower, than themonovalent affinity of a murine antibody (e.g., affinity of the murineantibody as a Fab fragment to human c-met) comprising, consisting orconsisting essentially of a light chain and heavy chain variable domainsequence as depicted in FIG. 7 (SEQ ID NO: 9 and 10). In anotherembodiment, the invention provides an anti-c-met humanized antibodywherein the monovalent affinity of the antibody to human c-met (e.g.,affinity of the antibody as a Fab fragment to human c-met) is greater,for example at least 3, 5, 7, 10 or 13-fold greater, than the monovalentaffinity of a murine antibody (e.g., affinity of the murine antibody asa Fab fragment to human c-met) comprising, consisting or consistingessentially of a light chain and heavy chain variable domain sequence asdepicted in FIG. 7 (SEQ ID NO: 9 and 10). In one embodiment, themonovalent affinity of the murine antibody to human c-met issubstantially the same as the binding affinity of a Fab fragmentcomprising variable domain sequences of an antibody produced byhybridoma cell line deposited under American Type Culture CollectionAccession Number ATCC HB-11894 (hybridoma 1A3.3.13) or HB-11895(hybridoma 5D5.11.6). As is well-established in the art, bindingaffinity of a ligand to its receptor can be determined using any of avariety of assays, and expressed in terms of a variety of quantitativevalues. Accordingly, in one embodiment, the binding affinity isexpressed as Kd values and reflects intrinsic binding affinity (e.g.,with minimized avidity effects). Generally and preferably, bindingaffinity is measured in vitro, whether in a cell-free or cell-associatedsetting. As described in greater detail herein, fold difference inbinding affinity can be quantified in terms of the ratio of themonovalent binding affinity value of a humanized antibody (e.g., in Fabform) and the monovalent binding affinity value of areference/comparator antibody (e.g., in Fab form) (e.g., a murineantibody having donor hypervariable region sequences), wherein thebinding affinity values are determined under similar assay conditions.Thus, in one embodiment, the fold difference in binding affinity isdetermined as the ratio of the Kd values of the humanized antibody inFab form and said reference/comparator Fab antibody. For example, in oneembodiment, if an antibody of the invention (A) has an affinity that is“3-fold lower” than the affinity of a reference antibody (M), then ifthe Kd value for A is 3×, the Kd value of M would be lx, and the ratioof Kd of A to Kd of M would be 3:1. Conversely, in one embodiment, if anantibody of the invention (C) has an affinity that is “3-fold greater”than the affinity of a reference antibody (R), then if the Kd value forC is 1×, the Kd value of R would be 3×, and the ratio of Kd of C to Kdof R would be 1:3. Any of a number of assays known in the art, includingthose described herein, can be used to obtain binding affinitymeasurements, including, for example, Biacore, radioimmunoassay (RIA)and ELISA.

In one aspect, a HGF/c-met antagonist of the invention comprises ananti-c-met antibody comprising:

(a) at least one, two, three, four or five hypervariable region (HVR)sequences selected from the group consisting of:

(i) HVR-L1 comprising sequence A1-A17, wherein A1-A17 isKSSQSLLYTSSQKNYLA (SEQ ID NO:1)

(ii) HVR-L2 comprising sequence B1-B7, wherein B1-B7 is WASTRES (SEQ IDNO:2)

(iii) HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQYYAYPWT (SEQID NO:3)

(iv)) HVR-H1 comprising sequence D1-D10, wherein D1-D10 is GYTFTSYWLH(SEQ ID NO:4)

(v) HVR-H2 comprising sequence E1-E 18, wherein E1-E 18 isGMIDPSNSDTRFNPNFKD (SEQ ID NO:5) and

(vi) HVR-H3 comprising sequence F1-F11, wherein F1-F11 is XYGSYVSPLDY(SEQ ID NO:6) and X is not R; and (b) at least one variant HVR, whereinthe variant HVR sequence comprises modification of at least one residueof the sequence depicted in SEQ ID NOs:1, 2, 3, 4, 5 or 6. In oneembodiment, HVR-L1 of an antibody of the invention comprises thesequence of SEQ ID NO:1. In one embodiment, HVR-L2 of an antibody of theinvention comprises the sequence of SEQ ID NO:2. In one embodiment,HVR-L3 of an antibody of the invention comprises the sequence of SEQ IDNO:3. In one embodiment, HVR-H1 of an antibody of the inventioncomprises the sequence of SEQ ID NO:4. In one embodiment, HVR-H2 of anantibody of the invention comprises the sequence of SEQ ID NO:5. In oneembodiment, HVR-H3 of an antibody of the invention comprises thesequence of SEQ ID NO:6. In one embodiment, HVR-H3 comprises TYGSYVSPLDY(SEQ ID NO: 7). In one embodiment, HVR-H3 comprises SYGSYVSPLDY (SEQ IDNO: 8). In one embodiment, an antibody of the invention comprising thesesequences (in combination as described herein) is humanized or human.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or six HVRs, wherein each HVR comprises, consists orconsists essentially of a sequence selected from the group consisting ofSEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, and 8, and wherein SEQ ID NO:1corresponds to an HVR-L1, SEQ ID NO:2 corresponds to an HVR-L2, SEQ IDNO:3 corresponds to an HVR-L3, SEQ ID NO:4 corresponds to an HVR-H1, SEQID NO:5 corresponds to an HVR-H2, and SEQ ID NOs:6, 7 or 8 correspondsto an HVR-H3. In one embodiment, an antibody of the invention comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, HVR-H2, and HVR-H3, wherein each, inorder, comprises SEQ ID NO:1, 2, 3, 4, 5 and 7. In one embodiment, anantibody of the invention comprises HVR-L1, HVR-L2, HVR-L3, HVR-H1,HVR-H2, and HVR-H3, wherein each, in order, comprises SEQ ID NO:1, 2, 3,4, 5 and 8.

Variant HVRs in an antibody of the invention can have modifications ofone or more residues within the HVR. In one embodiment, a HVR-L2 variantcomprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combination of thefollowing positions: B1 (M or L), B2 (P, T, G or S), B3 (N, G, R or T),B4 (I, N or F), B5 (P, I, L or G), B6 (A, D, T or V) and B7 (R, I, M orG). In one embodiment, a HVR-H1 variant comprises 1-5 (1, 2, 3, 4 or 5)substitutions in any combination of the following positions: D3 (N, P,L, S, A, I), D5 (I, S or Y), D6 (G, D, T, K, R), D7 (F, H, R, S, T or V)and D9 (M or V). In one embodiment, a HVR-H2 variant comprises 1-4 (1,2, 3 or 4) substitutions in any combination of the following positions:E7 (Y), E9 (I), E10 (I), E14 (T or Q), E15 (D, K, S, T or V), E16 (L),E17 (E, H, N or D) and E18 (Y, E or H). In one embodiment, a HVR-H3variant comprises 1-5 (1, 2, 3, 4 or 5) substitutions in any combinationof the following positions: F1 (T, S), F3 (R, S, H, T, A, K), F4 (G), F6(R, F, M, T, E, K, A, L, W), F7 (L, I, T, R, K, V), F8 (S, A), F10 (Y,N) and F11 (Q, S, H, F). Letter(s) in parenthesis following eachposition indicates an illustrative substitution (i.e., replacement)amino acid; as would be evident to one skilled in the art, suitabilityof other amino acids as substitution amino acids in the contextdescribed herein can be routinely assessed using techniques known in theart and/or described herein. In one embodiment, a HVR-L1 comprises thesequence of SEQ ID NO:1. In one embodiment, F1 in a variant HVR-H3 is T.In one embodiment, F1 in a variant HVR-H3 is S. In one embodiment, F3 ina variant HVR-H3 is R. In one embodiment, F3 in a variant HVR-H3 is S.In one embodiment, F7 in a variant HVR-H3 is T. In one embodiment, anantibody of the invention comprises a variant HVR-H3 wherein F1 is T orS, F3 is R or S, and F7 is T.

In one embodiment, an antibody of the invention comprises a variantHVR-H3 wherein F1 is T, F3 is R and F7 is T. In one embodiment, anantibody of the invention comprises a variant HVR-H3 wherein F1 is S. Inone embodiment, an antibody of the invention comprises a variant HVR-H3wherein F1 is T, and F3 is R. In one embodiment, an antibody of theinvention comprises a variant HVR-H3 wherein F1 is S, F3 is R and F7 isT.

In one embodiment, an antibody of the invention comprises a variantHVR-H3 wherein F1 is T, F3 is S, F7 is T, and F8 is S. In oneembodiment, an antibody of the invention comprises a variant HVR-H3wherein F1 is T, F3 is S, F7 is T, and F8 is A. In some embodiments,said variant HVR-H3 antibody further comprises HVR-L1, HVR-L2, HVR-L3,HVR-H1 and HVR-H2 wherein each comprises, in order, the sequencedepicted in SEQ ID NOs:1, 2, 3, 4 and 5. In some embodiments, theseantibodies further comprise a human subgroup III heavy chain frameworkconsensus sequence. In one embodiment of these antibodies, the frameworkconsensus sequence comprises substitution at position 71, 73 and/or 78.In some embodiments of these antibodies, position 71 is A, 73 is Tand/or 78 is A. In one embodiment of these antibodies, these antibodiesfurther comprise a human I light chain framework consensus sequence.

In one embodiment, an antibody of the invention comprises a variantHVR-L2 wherein B6 is V. In some embodiments, said variant HVR-L2antibody further comprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3,wherein each comprises, in order, the sequence depicted in SEQ ID NOs:1,3, 4, 5 and 6. In some embodiments, said variant HVR-L2 antibody furthercomprises HVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein eachcomprises, in order, the sequence depicted in SEQ ID NOs:1, 3, 4, 5 and7. In some embodiments, said variant HVR-L2 antibody further comprisesHVR-L1, HVR-L3, HVR-H1, HVR-H2 and HVR-H3, wherein each comprises, inorder, the sequence depicted in SEQ ID NOs:1, 3, 4, 5 and 8. In someembodiments, these antibodies further comprise a human subgroup IIIheavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment of theseantibodies, these antibodies further comprise a human I light chainframework consensus sequence.

In one embodiment, an antibody of the invention comprises a variantHVR-H2 wherein E14 is T, E15 is K and E11 is E. In one embodiment, anantibody of the invention comprises a variant HVR-H2 wherein E11 is E.In some embodiments, said variant HVR-H3 antibody further comprisesHVR-L1, HVR-L2, HVR-L3, HVR-H1, and HVR-H3 wherein each comprises, inorder, the sequence depicted in SEQ ID NOs:1, 2, 3, 4 and 6. In someembodiments, said variant HVR-H2 antibody further comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein each comprises, in order,the sequence depicted in SEQ ID NOs:1, 2, 3, 4, and 7. In someembodiments, said variant HVR-H2 antibody further comprises HVR-L1,HVR-L2, HVR-L3, HVR-H1, and HVR-H3, wherein each comprises, in order,the sequence depicted in SEQ ID NOs:1, 2, 3, 4, and 8. In someembodiments, these antibodies further comprise a human subgroup IIIheavy chain framework consensus sequence. In one embodiment of theseantibodies, the framework consensus sequence comprises substitution atposition 71, 73 and/or 78. In some embodiments of these antibodies,position 71 is A, 73 is T and/or 78 is A. In one embodiment of theseantibodies, these antibodies further comprise a human I light chainframework consensus sequence.

In one aspect, the invention provides an antibody comprising one, two,three, four, five or all of the HVR sequences depicted in FIGS. 2, 3and/or 4 (SEQ ID NOs:56-163).

A therapeutic agent for use in a host subject preferably elicits littleto no immunogenic response against the agent in said subject. In oneembodiment, the invention provides such an agent. For example, in oneembodiment, the invention provides a humanized antibody that elicitsand/or is expected to elicit a human anti-mouse antibody response (HAMA)at a substantially reduced level compared to an antibody comprising thesequence of SEQ ID NO: 9 & 10 in a host subject. In another example, theinvention provides a humanized antibody that elicits and/or is expectedto elicit minimal or no human anti-mouse antibody response (HAMA). Inone example, an antibody of the invention elicits anti-mouse antibodyresponse that is at or less than a clinically-acceptable level.

A humanized antibody of the invention may comprise one or more humanand/or human consensus non-hypervariable region (e.g., framework)sequences in its heavy and/or light chain variable domain. In someembodiments, one or more additional modifications are present within thehuman and/or human consensus non-hypervariable region sequences. In oneembodiment, the heavy chain variable domain of an antibody of theinvention comprises a human consensus framework sequence, which in oneembodiment is the subgroup III consensus framework sequence. In oneembodiment, an antibody of the invention comprises a variant subgroupIII consensus framework sequence modified at least one amino acidposition. For example, in one embodiment, a variant subgroup IIIconsensus framework sequence may comprise a substitution at one or moreof positions 71, 73 and/or 78. In one embodiment, said substitution isR71A, N73T and/or N78A, in any combination thereof.

As is known in the art, and as described in greater detail hereinbelow,the amino acid position/boundary delineating a hypervariable region ofan antibody can vary, depending on the context and the variousdefinitions known in the art (as described below). Some positions withina variable domain may be viewed as hybrid hypervariable positions inthat these positions can be deemed to be within a hypervariable regionunder one set of criteria while being deemed to be outside ahypervariable region under a different set of criteria. One or more ofthese positions can also be found in extended hypervariable regions (asfurther defined below). The invention provides antibodies comprisingmodifications in these hybrid hypervariable positions. In oneembodiment, these hybrid hypervariable positions include one or more ofpositions 26-30, 33-35B, 47-49, 57-65, 93, 94 and 102 in a heavy chainvariable domain. In one embodiment, these hybrid hypervariable positionsinclude one or more of positions 24-29, 35-36, 46-49, 56 and 97 in alight chain variable domain. In one embodiment, an antibody of theinvention comprises a variant human subgroup consensus frameworksequence modified at one or more hybrid hypervariable positions. In oneembodiment, an antibody of the invention comprises a heavy chainvariable domain comprising a variant human subgroup III consensusframework sequence modified at one or more of positions 27-28, 30,33-35, 49, 57-65, 94 and 102. In one embodiment, the antibody comprisesa F27Y substitution. In one embodiment, the antibody comprises a T28N,P, L, S, A or I substitution. In one embodiment, the antibody comprisesa S30I, T or Y substitution. In one embodiment, the antibody comprises aA33W substitution. In one embodiment, the antibody comprises a M34L orM34V substitution. In one embodiment, the antibody comprises a S35Hsubstitution. In one embodiment, the antibody comprises a T57Isubstitution. In one embodiment, the antibody comprises a Y58Rsubstitution. In one embodiment, the antibody comprises a Y59Fsubstitution. In one embodiment, the antibody comprises a A60Nsubstitution. In one embodiment, the antibody comprises a D61P, T or Qsubstitution. In one embodiment, the antibody comprises a S62N, D, K, Tor V substitution. In one embodiment, the antibody comprises a V63F orV63L substitution. In one embodiment, the antibody comprises a K64E, H,N, D or Q substitution. In one embodiment, the antibody comprises aG65D, Y, E or H substitution. In one embodiment, the antibody comprisesa R94T or R94S substitution. In one embodiment, the antibody comprises aY102Q, S, H or F substitution. In one embodiment, an antibody of theinvention comprising said R94T or R94S modification further comprisesone or more modifications at position 96 and/or 100. In one embodiment,said modifications comprise a G96R and/or S100T substitution (i.e., inHVR-H3). In one embodiment, an antibody of the invention comprises alight chain variable domain comprising a variant human kappa subgroup Iconsensus framework sequence modified at one or more of positions 24,25, 29 and 56. In one embodiment, the antibody comprises a R24Ksubstitution. In one embodiment, the antibody comprises a A25Ssubstitution. In one embodiment, the antibody comprises a I29Qsubstitution. In one embodiment, the antibody comprises a S56R, I, M orG substitution.

In one embodiment, an antibody of the invention comprises a heavy chainvariable domain comprising a variant human subgroup III consensusframework sequence modified at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17 or all of positions 27-28, 30, 33-35, 49, 57-65, 94and 102. In one embodiment, modification is selected from the groupconsisting of F27Y, T28(N, P, L, S, A or I), S30(I, T or Y), A33W,M34(L,V), S35H, T57I, Y58R, Y59F, A60N, D61(P, T, Q), S62(N, D, K, T,V),V63(F,L), K64(E, H, N, D, Q), G65(D, Y, E, H), R94(T,S) and Y102(Q, S,H, F). In one embodiment, an antibody of the invention comprising saidR94T or R94S modification further comprises one or more modifications atposition 96 and/or 100. In one embodiment, said modifications comprise aG96R and/or S100T substitution (i.e., in HVR-H3).

In one embodiment, an antibody of the invention comprises a light chainvariable domain comprising a variant human kappa subgroup I consensusframework sequence modified at 1, 2, 3 or all of positions 24, 25, 29and 56. In one embodiment, modification is selected from the groupconsisting of R24K, A25S, I29Q and S56(R, I, M, G).

An antibody of the invention can comprise any suitable human or humanconsensus light chain framework sequences, provided the antibodyexhibits the desired biological characteristics (e.g., a desired bindingaffinity). In one embodiment, an antibody of the invention comprises atleast a portion (or all) of the framework sequence of human light chain.In one embodiment, an antibody of the invention comprises at least aportion (or all) of human subgroup I framework consensus sequence.

In one embodiment, an antibody of the invention comprises a heavy and/orlight chain variable domain comprising framework sequence depicted inSEQ ID NO: 12 and/or 14 (FIG. 1), provided position 94 in the heavychain is not R (and is preferably but not necessarily S or T).

In one aspect, an antibody of the invention is a humanized anti-c-metantibody that inhibits binding of human hepatocyte growth factor to itsreceptor better than a reference antibody comprising a chimericanti-c-met antibody comprising a light chain and heavy chain variablesequence as depicted in FIG. 7 (SEQ ID NO: 9 and 10). For example, inone embodiment, an antibody of the invention inhibits HGF binding withan IC50 value that is less than about half that of the chimericantibody. In one embodiment, the IC50 value of an antibody of theinvention is about 0.1, 0.2, 0.3 or 0.4 that of the chimeric antibody.Comparison of abilities to inhibit HGF binding to its receptor can beperformed according to various methods known in the art, including asdescribed in the Examples below. In one embodiment, IC50 values aredetermined across an antibody concentration range from about 0.01 nM toaround 1000 nM.

In one aspect, an antibody of the invention is a humanized anti-c-metantibody that inhibits human hepatocyte growth factor (HGF) receptoractivation better than a reference antibody comprising a chimericanti-c-met antibody comprising a light chain and heavy chain variablesequence as depicted in FIG. 7 (SEQ ID NO: 9 and 10). For example in oneembodiment, an antibody of the invention inhibits receptor activationwith an IC50 value that is less than about half that of the chimericantibody. In one embodiment, the IC50 value of an antibody of theinvention is about 0.1, 0.2, 0.3 or 0.4 that of the chimeric antibody.Comparison of abilities to inhibit HGF receptor activation can beperformed according to various methods known in the art, including asdescribed in the Examples below. In one embodiment, IC50 values aredetermined across an antibody concentration range from about 0.1 nM toabout 100 nM.

In one aspect, an antibody of the invention is a humanized anti-c-metantibody that inhibits c-met-dependent cell proliferation better than areference antibody comprising a chimeric anti-c-met antibody comprisinga light chain and heavy chain variable sequence as depicted in FIG. 7(SEQ ID NO: 9 and 10). For example, in one embodiment, an antibody ofthe invention inhibits cell proliferation with an IC50 value that isless than about half that of the chimeric antibody. In one embodiment,the IC50 value of an antibody of the invention is about 0.1, 0.2, 0.3 or0.4 that of the chimeric antibody. Comparison of abilities to inhibitcell proliferation can be performed according to various methods knownin the art, including as described in the Examples below. In oneembodiment, IC50 values are determined across an antibody concentrationrange from about 0.01 nM to about 100 nM.

In one embodiment, both the humanized antibody and chimeric antibody aremonovalent. In one embodiment, both the humanized antibody and chimericantibody comprise a single Fab region linked to an Fc region. In oneembodiment, the reference chimeric antibody comprises variable domainsequences depicted in FIG. 7 (SEQ ID NO: 9 and 10) linked to a human Fcregion. In one embodiment, the human Fc region is that of an IgG (e.g.,IgG1, 2, 3 or 4).

In one aspect, the invention provides an antibody comprising a heavychain variable domain comprising the HVR1-HC, HVR2-HC and/or HVR3-HCsequence depicted in FIG. 13. In one embodiment, the variable domaincomprises FR1-HC, FR2-HC, FR3-HC and/or FR4-HC sequence depicted in FIG.13. In one embodiment, the antibody comprises CH1 and/or Fc sequencedepicted in FIG. 13. In one embodiment, an antibody of the inventioncomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence depicted in FIG. 13. In one embodiment, an antibody of theinvention comprises a heavy chain variable domain comprising theHVR1-HC, HVR2-HC and/or HVR3-HC sequence, and the CH1 and/or Fc sequencedepicted in FIG. 13. In one embodiment, an antibody of the inventioncomprises a heavy chain variable domain comprising the HVR1-HC, HVR2-HCand/or HVR3-HC sequence, and the FR1-HC, FR2-HC, FR3-HC and/or FR4-HCsequence depicted in FIG. 13, and the CH1 and/or Fc sequence depicted inFIG. 13. In one embodiment, the Fc region of the antibody of theinvention comprises a complex between a polypeptide comprising the Fcsequence in FIG. 13 and a polypeptide comprising the Fc sequence in FIG.14.

In one aspect, the invention provides an antibody comprising a lightchain variable domain comprising HVR1-LC, HVR2-LC and/or HVR3-LCsequence depicted in FIG. 13. In one embodiment, the variable domaincomprises FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence depicted in FIG.13. In one embodiment, the antibody comprises CL1 sequence depicted inFIG. 13.

In one embodiment, an antibody of the invention comprises light andheavy chain variable domains as described in the preceding twoparagraphs. In one embodiment, the antibody is monovalent and comprisesan Fc region. In one embodiment, the Fc region comprises at least oneprotuberance (knob) and at least one cavity (hole), wherein presence ofthe protuberance and cavity enhances formation of a complex between anFc polypeptide comprising the protuberance and an Fc polypeptidecomprising the cavity, for example as described in WO 2005/063816. Inone embodiment, the Fc region of an antibody of the invention comprisesa first and a second Fc polypeptide, wherein the first and secondpolypeptide each comprises one or more mutations with respect to wildtype human Fc. In one embodiment, a cavity mutation is T366S, L368Aand/or Y407V. In one embodiment, a protuberance mutation is T366W. Inone embodiment, the first polypeptide comprises the Fc sequence depictedin FIG. 13 and the second polypeptide comprises the Fc sequence depictedin FIG. 14.

Antagonists of the invention can be used to modulate one or more aspectsof HGF/c-met-associated effects, including but not limited to c-metactivation, downstream molecular signaling (e.g., mitogen activatedprotein kinase (MAPK) phosphorylation), cell proliferation, cellmigration, cell survival, cell morphogenesis and angiogenesis. Theseeffects can be modulated by any biologically relevant mechanism,including disruption of ligand (e.g., HGF) binding to c-met, c-metphosphorylation and/or c-met multimerization. Accordingly, in oneembodiment, the invention provides a c-met antagonist antibody thatinhibits binding of HGF to c-met. In one embodiment, a c-met antagonistantibody of the invention disrupts c-met multimerization (e.g.,dimerization). In one embodiment, a c-met antagonist antibody of theinvention disrupts dimerization function of c-met Sema domain. In oneexample, a c-met antagonist antibody interferes with ability of c-metSema domain to effect c-met dimerization. Interference can be direct orindirect. For example, a c-met antagonist antibody may bind to asequence within the c-met Sema domain, and thereby inhibit interactionof said bound domain with its binding partner (such as another c-metmolecule). In another example, a c-met antagonist antibody may bind to asequence that is not within the c-met Sema domain, but wherein saidbinding results in disruption of the ability of the c-met Sema domain tointeract with its binding partner (such as another c-met molecule). Inone embodiment, an antagonist antibody of the invention binds to c-met(e.g., the extracellular domain) such that c-met dimerization isdisrupted. In one embodiment, an antagonist antibody of the inventionbinds to c-met such that ability of c-met Sema domain to effect c-metdimerization is disrupted. For example, in one embodiment, the inventionprovides an antagonist antibody which upon binding to a c-met moleculeinhibits dimerization of said molecule. In one embodiment, a c-metantagonist antibody of the invention specifically binds a sequence inthe c-met Sema domain.

In one embodiment, an antagonist antibody of the invention disruptsc-met dimerization comprising homodimerization. In one embodiment, anantagonist antibody of the invention disrupts c-met dimerizationcomprising heterodimerization (i.e., c-met dimerization with a non-c-metmolecule).

In some instances, it may be advantageous to have a c-met antagonistantibody that does not interfere with binding of a ligand (such as HGF)to c-met. Accordingly, in one embodiment, the invention provides anantibody that does not bind an HGF binding site on c-met. In anotherembodiment, an antibody of the invention does not substantially inhibitHGF binding to c-met. In one embodiment, an antibody of the inventiondoes not substantially compete with HGF for binding to c-met. In oneexample, an antagonist antibody of the invention can be used inconjunction with one or more other antagonists, wherein the antagonistsare targeted at different processes and/or functions within theHGF/c-met axis. Thus, in one embodiment, a c-met antagonist antibody ofthe invention binds to an epitope on c-met distinct from an epitopebound by another c-met antagonist (such as the Fab fragment of themonoclonal antibody produced by the hybridoma cell line deposited underAmerican Type Culture Collection Accession Number ATCC HB-11894(hybridoma 1A3.3.13)). In another embodiment, a c-met antagonistantibody of the invention is distinct from (i.e., it is not) a Fabfragment of the monoclonal antibody produced by the hybridoma cell linedeposited under American Type Culture Collection Accession Number ATCCHB-11894 (hybridoma 1A3.3.13).

In one embodiment, the invention provides a c-met antagonist antibodythat disrupts both c-met multimerization and ligand binding. Forexample, an antagonist antibody of the invention that inhibits c-metmultimerization (e.g., dimerization) may further comprise an ability tocompete with HGF for binding to c-met.

In one embodiment of a c-met antagonist antibody of the invention,binding of the antagonist to c-met inhibits c-met activation by HGF. Inanother embodiment of a c-met antagonist antibody of the invention,binding of the antagonist to c-met in a cell inhibits proliferation,survival, scattering, morphogenesis and/or motility of the cell.

In one embodiment, a c-met antagonist antibody of the inventionspecifically binds at least a portion of c-met Sema domain or variantthereof. In one example, an antagonist antibody of the inventionspecifically binds at least one of the sequences selected from the groupconsisting of LDAQT (SEQ ID NO:15) (e.g., residues 269-273 of c-met),LTEKRKKRS (SEQ ID NO:16) (e.g., residues 300-308 of c-met), KPDSAEPM(SEQ ID NO:17) (e.g., residues 350-357 of c-met) and NVRCLQHF (SEQ IDNO:18) (e.g., residues 381-388 of c-met). In one embodiment, anantagonist antibody of the invention specifically binds a conformationalepitope formed by part or all of at least one of the sequences selectedfrom the group consisting of LDAQT (e.g., residues 269-273 of c-met),LTEKRKKRS (e.g., residues 300-308 of c-met), KPDSAEPM (e.g., residues350-357 of c-met) and NVRCLQHF (e.g., residues 381-388 of c-met). In oneembodiment, an antagonist antibody of the invention specifically bindsan amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 95%, 98%sequence identity or similarity with the sequence LDAQT, LTEKRKKRS,KPDSAEPM and/or NVRCLQHF.

In one embodiment, an antibody of the invention specifically binds toHGF receptor of a first animal species, and does not specifically bindto HGF receptor of a second animal species. In one embodiment, the firstanimal species is human and/or primate (e.g., cynomolgus monkey), andthe second animal species is murine (e.g., mouse) and/or canine 1n oneembodiment, the first animal species is human. In one embodiment, thefirst animal species is primate, for example cynomolgus monkey. In oneembodiment, the second animal species is murine, for example mouse. Inone embodiment, the second animal species is canine

In one aspect, the invention provides compositions comprising one ormore antagonist antibodies of the invention and a carrier. In oneembodiment, the carrier is pharmaceutically acceptable.

In one aspect, the invention provides nucleic acids encoding a c-metantagonist antibody of the invention.

In one aspect, the invention provides vectors comprising a nucleic acidof the invention.

In one aspect, the invention provides host cells comprising a nucleicacid or a vector of the invention. A vector can be of any type, forexample a recombinant vector such as an expression vector. Any of avariety of host cells can be used. In one embodiment, a host cell is aprokaryotic cell, for example, E. coli. In one embodiment, a host cellis a eukaryotic cell, for example a mammalian cell such as ChineseHamster Ovary (CHO) cell.

In one aspect, the invention provides methods for making an antagonistof the invention. For example, the invention provides a method of makinga c-met antagonist antibody (which, as defined herein includes fulllength and fragments thereof), said method comprising expressing in asuitable host cell a recombinant vector of the invention encoding saidantibody (or fragment thereof), and recovering said antibody.

In one aspect, the invention provides an article of manufacturecomprising a container; and a composition contained within thecontainer, wherein the composition comprises one or more c-metantagonist antibodies of the invention. In one embodiment, thecomposition comprises a nucleic acid of the invention. In oneembodiment, a composition comprising an antagonist antibody furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, an article of manufacture of theinvention further comprises instructions for administering thecomposition (e.g., the antagonist antibody) to a subject.

In one aspect, the invention provides a kit comprising a first containercomprising a composition comprising one or more c-met antagonistantibodies of the invention; and a second container comprising a buffer.In one embodiment, the buffer is pharmaceutically acceptable. In oneembodiment, a composition comprising an antagonist antibody furthercomprises a carrier, which in some embodiments is pharmaceuticallyacceptable. In one embodiment, a kit further comprises instructions foradministering the composition (e.g., the antagonist antibody) to asubject.

In one aspect, the invention provides use of a c-met antagonist antibodyof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumor, acell proliferative disorder, an immune (such as autoimmune) disorderand/or an angiogenesis-related disorder.

In one aspect, the invention provides use of a nucleic acid of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an expression vector of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of a host cell of theinvention in the preparation of a medicament for the therapeutic and/orprophylactic treatment of a disease, such as a cancer, a tumor, a cellproliferative disorder, an immune (such as autoimmune) disorder and/oran angiogenesis-related disorder.

In one aspect, the invention provides use of an article of manufactureof the invention in the preparation of a medicament for the therapeuticand/or prophylactic treatment of a disease, such as a cancer, a tumor, acell proliferative disorder, an immune (such as autoimmune) disorderand/or an angiogenesis-related disorder.

In one aspect, the invention provides use of a kit of the invention inthe preparation of a medicament for the therapeutic and/or prophylactictreatment of a disease, such as a cancer, a tumor, a cell proliferativedisorder, an immune (such as autoimmune) disorder and/or anangiogenesis-related disorder.

The invention provides methods and compositions useful for modulatingdisease states associated with dysregulation of the HGF/c-met signalingaxis. The HGF/c-met signaling pathway is involved in multiple biologicaland physiological functions, including, e.g., cell proliferation andangiogenesis. Thus, in one aspect, the invention provides a methodcomprising administering to a subject an antibody of the invention.

In one aspect, the invention provides a method of inhibiting c-metactivated cell proliferation, said method comprising contacting a cellor tissue with an effective amount of a antibody of the invention,whereby cell proliferation associated with c-met activation isinhibited.

In one aspect, the invention provides a method of treating apathological condition associated with dysregulation of c-met activationin a subject, said method comprising administering to the subject aneffective amount of an antibody of the invention, whereby said conditionis treated.

In one aspect, the invention provides a method of inhibiting the growthof a cell that expresses c-met or hepatocyte growth factor, or both,said method comprising contacting said cell with an antibody of theinvention thereby causing an inhibition of growth of said cell. In oneembodiment, the cell is contacted by HGF expressed by a different cell(e.g., through a paracrine effect).

In one aspect, the invention provides a method of therapeuticallytreating a mammal having a cancerous tumor comprising a cell thatexpresses c-met or hepatocyte growth factor, or both, said methodcomprising administering to said mammal an effective amount of anantibody of the invention, thereby effectively treating said mammal. Inone embodiment, the cell is contacted by HGF expressed by a differentcell (e.g., through a paracrine effect).

In one aspect, the invention provides a method for treating orpreventing a cell proliferative disorder associated with increasedexpression or activity of c-met or hepatocyte growth, or both, saidmethod comprising administering to a subject in need of such treatmentan effective amount of an antibody of the invention, thereby effectivelytreating or preventing said cell proliferative disorder. In oneembodiment, said proliferative disorder is cancer.

In one aspect, the invention provides a method for inhibiting the growthof a cell, wherein growth of said cell is at least in part dependentupon a growth potentiating effect of c-met or hepatocyte growth factor,or both, said method comprising contacting said cell with an effectiveamount of an antibody of the invention, thereby inhibiting the growth ofsaid cell. In one embodiment, the cell is contacted by HGF expressed bya different cell (e.g., through a paracrine effect).

A method of therapeutically treating a tumor in a mammal, wherein thegrowth of said tumor is at least in part dependent upon a growthpotentiating effect of c-met or hepatocyte growth factor, or both, saidmethod comprising contacting said cell with an effective amount of anantibody of the invention, thereby effectively treating said tumor. Inone embodiment, the cell is contacted by HGF expressed by a differentcell (e.g., through a paracrine effect).

Methods of the invention can be used to affect any suitable pathologicalstate, for example, cells and/or tissues associated with dysregulationof the HGF/c-met signaling pathway. In one embodiment, a cell that istargeted in a method of the invention is a cancer cell. For example, acancer cell can be one selected from the group consisting of a breastcancer cell, a colorectal cancer cell, a lung cancer cell, a papillarycarcinoma cell (e.g., of the thyroid gland), a colon cancer cell, apancreatic cancer cell, an ovarian cancer cell, a cervical cancer cell,a central nervous system cancer cell, an osteogenic sarcoma cell, arenal carcinoma cell, a hepatocellular carcinoma cell, a bladder cancercell, a gastric carcinoma cell, a head and neck squamous carcinoma cell,a melanoma cell and a leukemia cell. In one embodiment, a cell that istargeted in a method of the invention is a hyperproliferative and/orhyperplastic cell. In one embodiment, a cell that is targeted in amethod of the invention is a dysplastic cell. In yet another embodiment,a cell that is targeted in a method of the invention is a metastaticcell.

Methods of the invention can further comprise additional treatmentsteps. For example, in one embodiment, a method further comprises a stepwherein a targeted cell and/or tissue (e.g., a cancer cell) is exposedto radiation treatment or a chemotherapeutic agent.

As described herein, c-met activation is an important biological processthe dysregulation of which leads to numerous pathological conditions.Accordingly, in one embodiment of methods of the invention, a cell thatis targeted (e.g., a cancer cell) is one in which activation of c-met isenhanced as compared to a normal cell of the same tissue origin. In oneembodiment, a method of the invention causes the death of a targetedcell. For example, contact with an antagonist of the invention mayresult in a cell's inability to signal through the c-met pathway, whichresults in cell death.

Dysregulation of c-met activation (and thus signaling) can result from anumber of cellular changes, including, for example, overexpression ofHGF (c-met's cognate ligand) and/or c-met itself. Accordingly, in someembodiments, a method of the invention comprises targeting a cellwherein c-met or hepatoctye growth factor, or both, is more abundantlyexpressed by said cell (e.g., a cancer cell) as compared to a normalcell of the same tissue origin. A c-met-expressing cell can be regulatedby HGF from a variety of sources, i.e. in an autocrine or paracrinemanner. For example, in one embodiment of methods of the invention, atargeted cell is contacted/bound by hepatocyte growth factor expressedin a different cell (e.g., via a paracrine effect). Said different cellcan be of the same or of a different tissue origin. In one embodiment, atargeted cell is contacted/bound by HGF expressed by the targeted cellitself (e.g., via an autocrine effect/loop). C-met activation and/orsignaling can also occur independent of ligand. Hence, in one embodimentof methods of the invention, c-met activation in a targeted cell occursindependent of ligand.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B depict alignment of sequences of the variable light andheavy chains for the following: light chain human subgroup I consensussequence, heavy chain human subgroup III consensus sequence, murine 5D5anti-c-met antibody and 5D5-grafted “humanized” antibody.

FIGS. 2A and 2B depict various HVR sequences of selectedaffinity-matured antibodies from libraries with individually-randomizedHVR.

FIG. 3 depicts HVR-H3 sequences of selected affinity-matured antibodiesfrom a library pool comprising a combination of 6 libraries encompassingall six HVRs wherein each library is randomized in a single HVR.

FIG. 4 depicts results of Biacore analysis of selected anti-c-metantibodies.

FIGS. 5A,B & 6A,B depict exemplary acceptor human consensus frameworksequences for use in practicing the instant invention with sequenceidentifiers as follows:

-   -   Variable heavy (VH) consensus frameworks (FIG. 5A, B)        human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID        NO:19)        human VH subgroup I consensus framework minus extended        hypervariable regions (SEQ ID NOs:20-22)        human VH subgroup II consensus framework minus Kabat CDRs (SEQ        ID NO:23)        human VH subgroup II consensus framework minus extended        hypervariable regions (SEQ ID NOs:24-26)        human VH subgroup III consensus framework minus Kabat CDRs (SEQ        ID NO:27)        human VH subgroup III consensus framework minus extended        hypervariable regions (SEQ ID NOs:28-30)        human VH acceptor framework minus Kabat CDRs (SEQ ID NO:31)        human VH acceptor framework minus extended hypervariable regions        (SEQ ID NOs:32-33)        human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:34)        human VH acceptor 2 framework minus extended hypervariable        regions (SEQ ID NOs:35-37)    -   Variable light (VL) consensus frameworks (FIG. 6A,B)        human VL kappa subgroup I consensus framework (SEQ ID NO:38)        human VL kappa subgroup II consensus framework (SEQ ID NO:39)        human VL kappa subgroup III consensus framework (SEQ ID NO:40)        human VL kappa subgroup IV consensus framework (SEQ ID NO:41)

FIG. 7 depicts donor (murine antibody 5D5) light chain (LC) and heavychain (HC) variable domain sequences.

FIG. 8 depicts graphical data for blocking of HGF binding to itsreceptor by an antibody of the invention.

FIG. 9 depicts graphical data for inhibition of HGF receptor activationby an antibody of the invention.

FIG. 10 depicts graphical data for inhibition of cell proliferation byan antibody of the invention. “rchOA5D5 HGF” refers to chimericone-armed 5D5 antibody plus HGF; “rhuOA5D5v2 HGF” refers to OA5D5.v2plus HGF; “rhuOA5D5v1 HGF” refers to OA5D5.v1 plus HGF”. “rchOA5D5Control” refers to chimeric one-armed 5D5 antibody without HGF;“rhuOA5D5v2 Control” refers to OA5D5.v2 without HGF; “rhuOA5D5v1Control” refers to OA5D5.v1 without HGF”.

FIG. 11A, B depicts data for inhibition of receptor phosphorylation inthe presence of an antibody of the invention. FIG. 11A depicts receptorphosphorylation of H358 cells. FIG. 11B depicts receptor phosphorylationof H358 cells transfected with HGF.

FIG. 12 depicts graphical data showing in vivo efficacy of an antibodyof the invention. “TI” refers to tumor incidence. TI=8/10 refers to 8mice having tumors out of a group of 10 mice. TI=2/8 refers to 2 micehaving tumors out of a group of 8 mice.

FIG. 13 depicts amino acid sequences of the framework (FR),hypervariable region (HVR), first constant domain (CL or CH1) and Fcregion (Fc) of one embodiment of an antibody of the invention (5D5.v2).The Fc sequence depicted comprises “hole” (cavity) mutations T366S,L368A and Y407V, as described in WO 2005/063816.

FIG. 14 depicts sequence of an Fc polypeptide comprising “knob”(protuberance) mutation T366W, as described in WO 2005/063816. In oneembodiment, an Fc polypeptide comprising this sequence forms a complexwith an Fc polypeptide comprising the Fc sequence of FIG. 13 to generatean Fc region of an antibody of the invention.

MODES FOR CARRYING OUT THE INVENTION

The invention provides methods, compositions, kits and articles ofmanufacture for identifying and/or using inhibitors of the HGF/c-metsignaling pathway.

Details of these methods, compositions, kits and articles of manufactureare provided herein.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry, andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, “Molecular Cloning: ALaboratory Manual”, second edition (Sambrook et al., 1989);“Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “Animal CellCulture” (R. I. Freshney, ed., 1987); “Methods in Enzymology” (AcademicPress, Inc.); “Current Protocols in Molecular Biology” (F. M. Ausubel etal., eds., 1987, and periodic updates); “PCR: The Polymerase ChainReaction”, (Mullis et al., ed., 1994); “A Practical Guide to MolecularCloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual”(Barbas et al., 2001).

DEFINITIONS

A “modification” of an amino acid residue/position, as used herein,refers to a change of a primary amino acid sequence as compared to astarting amino acid sequence, wherein the change results from a sequencealteration involving said amino acid residue/positions. For example,typical modifications include substitution of the residue (or at saidposition) with another amino acid (e.g., a conservative ornon-conservative substitution), insertion of one or more (generallyfewer than 5 or 3) amino acids adjacent to said residue/position, anddeletion of said residue/position. An “amino acid substitution”, orvariation thereof, refers to the replacement of an existing amino acidresidue in a predetermined (starting) amino acid sequence with adifferent amino acid residue. Generally and preferably, the modificationresults in alteration in at least one physicobiochemical activity of thevariant polypeptide compared to a polypeptide comprising the starting(or “wild type”) amino acid sequence. For example, in the case of anantibody, a physicobiochemical activity that is altered can be bindingaffinity, binding capability and/or binding effect upon a targetmolecule.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat”, and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, a FR or CDR of the variable domain. For example, a heavychain variable domain may include a single amino acid insert (residue52a according to Kabat) after residue 52 of H2 and inserted residues(e.g. residues 82a, 82b, and 82c, etc according to Kabat) after heavychain FR residue 82. The Kabat numbering of residues may be determinedfor a given antibody by alignment at regions of homology of the sequenceof the antibody with a “standard” Kabat numbered sequence.

The phrase “substantially similar,” or “substantially the same”, as usedherein, denotes a sufficiently high degree of similarity between twonumeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of little or no biological and/or statisticalsignificance within the context of the biological characteristicmeasured by said values (e.g., Kd values). The difference between saidtwo values is preferably less than about 50%, preferably less than about40%, preferably less than about 30%, preferably less than about 20%,preferably less than about 10% as a function of the value for thereference/comparator antibody.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

In one embodiment, the “Kd” or “Kd value” according to this invention ismeasured by a radiolabeled antigen binding assay (RIA) performed withthe Fab version of an antibody of interest and its antigen as describedby the following assay that measures solution binding affinity of Fabsfor antigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 ug/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (e.g., consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature (e.g., for one hour).The solution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodimentthe Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

An “on-rate” or “rate of association” or “association rate” or “k_(on)”according to this invention can also be determined with the same surfaceplasmon resonance technique described above using a BIAcore™-2000 or aBIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilizedantigen CM5 chips at ˜10 response units (RU). Briefly, carboxymethylateddextran biosensor chips (CM5, BIAcore Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, into 5 ug/ml (˜0.2uM) before injection at a flow rate of 5 ul/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of 1M ethanolamine to block unreacted groups. For kineticsmeasurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) areinjected in PBS with 0.05% Tween 20 (PBST) at 25° C. at a flow rate ofapproximately 25 ul/min. Association rates (k_(on)) and dissociationrates (k_(off)) are calculated using a simple one-to-one Langmuirbinding model (BIAcore Evaluation Software version 3.2) by simultaneousfitting the association and dissociation sensorgram. The equilibriumdissociation constant (Kd) was calculated as the ratio k_(off)/k_(on).See, e.g., Chen, Y., et al., (1999) J. Mol. Biol 293:865-881. However,if the on-rate exceeds 10⁶ M⁻¹ S⁻¹ by the surface plasmon resonanceassay above, then the on-rate is preferably determined by using afluorescent quenching technique that measures the increase or decreasein fluorescence emission intensity (excitation=295 nm; emission=340 nm,16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form)in PBS, pH 7.2, in the presence of increasing concentrations of antigenas measured in a spectrometer, such as a stop-flow equippedspectrophometer (Aviv Instruments) or a 8000-series SLM-Amincospectrophotometer (ThermoSpectronic) with a stirred cuvette. The “Kd” or“Kd value” according to this invention is in one embodiment measured bya radiolabeled antigen binding assay (RIA) performed with the Fabversion of the antibody and antigen molecule as described by thefollowing assay that measures solution binding affinity of Fabs forantigen by equilibrating Fab with a minimal concentration of(¹²⁵I)-labeled antigen in the presence of a titration series ofunlabeled antigen, then capturing bound antigen with an anti-Fabantibody-coated plate (Chen, et al., (1999) J. Mol. Biol 293:865-881).To establish conditions for the assay, microtiter plates (Dynex) arecoated overnight with 5 ug/ml of a capturing anti-Fab antibody (CappelLabs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with2% (w/v) bovine serum albumin in PBS for two to five hours at roomtemperature (approximately 23° C.). In a non-adsorbant plate (Nunc#269620), 100 pM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutionsof a Fab of interest (consistent with assessment of an anti-VEGFantibody, Fab-12, in Presta et al., (1997) Cancer Res. 57:4593-4599).The Fab of interest is then incubated overnight; however, the incubationmay continue for a longer period (e.g., 65 hours) to insure thatequilibrium is reached. Thereafter, the mixtures are transferred to thecapture plate for incubation at room temperature for one hour. Thesolution is then removed and the plate washed eight times with 0.1%Tween-20 in PBS. When the plates have dried, 150 ul/well of scintillant(MicroScint-20; Packard) is added, and the plates are counted on aTopcount gamma counter (Packard) for ten minutes. Concentrations of eachFab that give less than or equal to 20% of maximal binding are chosenfor use in competitive binding assays. According to another embodiment,the Kd or Kd value is measured by using surface plasmon resonance assaysusing a BIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway,N.J.) at 25 C with immobilized antigen CM5 chips at ˜10 response units(RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcoreInc.) are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)is calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol. 293:865-881. If the on-rate exceeds 10⁶ M⁻¹ S⁻¹ bythe surface plasmon resonance assay above, then the on-rate can bedetermined by using a fluorescent quenching technique that measures theincrease or decrease in fluorescence emission intensity (excitation=295nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigenantibody (Fab form) in PBS, pH 7.2, in the presence of increasingconcentrations of antigen as measured in a spectrometer, such as astop-flow equipped spectrophometer (Aviv Instruments) or a 8000-seriesSLM-Aminco spectrophotometer (ThermoSpectronic) with a stirred cuvette.

In one embodiment, an “on-rate” or “rate of association” or “associationrate” or “k_(on)” according to this invention is determined with thesame surface plasmon resonance technique described above using aBIAcore™-2000 or a BIAcore™-3000 (BIAcore, Inc., Piscataway, N.J.) at 25C with immobilized antigen CM5 chips at ˜10 response units (RU).Briefly, carboxymethylated dextran biosensor chips (CM5, BIAcore Inc.)are activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimidehydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to thesupplier's instructions. Antigen is diluted with 10 mM sodium acetate,pH 4.8, into 5 ug/ml (˜0.2 uM) before injection at a flow rate of 5ul/minute to achieve approximately 10 response units (RU) of coupledprotein. Following the injection of antigen, 1M ethanolamine is injectedto block unreacted groups. For kinetics measurements, two-fold serialdilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05%Tween 20 (PBST) at 25° C. at a flow rate of approximately 25 ul/min.Association rates (k_(on)) and dissociation rates (k_(off)) arecalculated using a simple one-to-one Langmuir binding model (BIAcoreEvaluation Software version 3.2) by simultaneous fitting the associationand dissociation sensorgram. The equilibrium dissociation constant (Kd)was calculated as the ratio k_(off)/k_(on). See, e.g., Chen, Y., et al.,(1999) J. Mol. Biol 293:865-881. However, if the on-rate exceeds 10⁶ M⁻¹S⁻¹ by the surface plasmon resonance assay above, then the on-rate ispreferably determined by using a fluorescent quenching technique thatmeasures the increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophometer (Aviv Instruments) or a8000-series SLM-Aminco spectrophotometer (ThermoSpectronic) with astirred cuvette.

The phrase “substantially reduced,” or “substantially different”, asused herein, denotes a sufficiently high degree of difference betweentwo numeric values (generally one associated with an antibody of theinvention and the other associated with a reference/comparator antibody)such that one of skill in the art would consider the difference betweenthe two values to be of statistical significance within the context ofthe biological characteristic measured by said values (e.g., Kd values,HAMA response). The difference between said two values is preferablygreater than about 10%, preferably greater than about 20%, preferablygreater than about 30%, preferably greater than about 40%, preferablygreater than about 50% as a function of the value for thereference/comparator antibody.

“Percent (%) amino acid sequence identity” with respect to a peptide orpolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the specific peptide or polypeptide sequence, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Alignment for purposesof determining percent amino acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. Those skilled in the art can determineappropriate parameters for measuring alignment, including any algorithmsneeded to achieve maximal alignment over the full length of thesequences being compared. For purposes herein, however, % amino acidsequence identity values are generated using the sequence comparisoncomputer program ALIGN-2, wherein the complete source code for theALIGN-2 program is provided in Table A below. The ALIGN-2 sequencecomparison computer program was authored by Genentech, Inc. and thesource code shown in Table A below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin FIG. 8 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

where X is the number of amino acid residues scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofA and B, and where Y is the total number of amino acid residues in B. Itwill be appreciated that where the length of amino acid sequence A isnot equal to the length of amino acid sequence B, the % amino acidsequence identity of A to B will not equal the % amino acid sequenceidentity of B to A.

Unless specifically stated otherwise, all % amino acid sequence identityvalues used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program.

TABLE A /*  *  * C-C increased from 12 to 15  * Z is average of EQ  * Bis average of ND  * match with stop is _M; stop-stop = 0; J (joker)match = 0  */ #define _M −8 /* value of a match with a stop */ int_day[26][26] = { /* A B C D E F G H I J K L M N O P Q R S T U V W X Y Z*/ /* A */ { 2, 0,−2, 0, 0,−4, 1,−1,−1, 0,−1,−2,−1, 0,_M, 1, 0,−2, 1, 1,0, 0,−6, 0,−3, 0}, /* B */ { 0, 3,−4, 3, 2,−5, 0, 1,−2, 0, 0,−3,−2,2,_M,−1, 1, 0, 0, 0, 0,−2,−5, 0,−3, 1}, /* C */{−2,−4,15,−5,−5,−4,−3,−3,−2, 0,−5,−6,−5,−4,_M,−3,−5,−4, 0,−2, 0,−2,−8,0, 0,−5}, /* D */ { 0, 3,−5, 4, 3,−6, 1, 1,−2, 0, 0,−4,−3, 2,_M,−1,2,−1, 0, 0, 0,−2,−7, 0,−4, 2}, /* E */ { 0, 2,−5, 3, 4,−5, 0, 1,−2, 0,0,−3,−2, 1,_M,−1, 2,−1, 0, 0, 0,−2,−7, 0,−4, 3}, /* F */{−4,−5,−4,−6,−5, 9,−5,−2, 1, 0,−5, 2, 0,−4,_M,−5,−5,−4,−3,−3, 0,−1, 0,0, 7,−5}, /* G */ { 1, 0,−3, 1, 0,−5, 5,−2,−3, 0,−2,−4,−3,0,_M,−1,−1,−3, 1, 0, 0,−1,−7, 0,−5, 0}, /* H */ {−1, 1,−3, 1, 1,−2,−2,6,−2, 0, 0,−2,−2, 2,_M, 0, 3, 2,−1,−1, 0,−2,−3, 0, 0, 2}, /* I */{−1,−2,−2,−2,−2, 1,−3,−2, 5, 0,−2, 2, 2,−2,_M,−2,−2,−2,−1, 0, 0, 4,−5,0,−1,−2}, /* J */ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0,−5, 0, 0,−5,−2, 0,−2, 0,5,−3, 0, 1,_M,−1, 1, 3, 0, 0, 0,−2,−3, 0,−4, 0}, /* L */{−2,−3,−6,−4,−3, 2,−4,−2, 2, 0,−3, 6, 4,−3,_M,−3,−2,−3,−3,−1, 0, 2,−2,0,−1,−2}, /* M */ {−1,−2,−5,−3,−2, 0,−3,−2, 2, 0, 0, 4, 6,−2,_M,−2,−1,0,−2,−1, 0, 2,−4, 0,−2,−1}, /* N */ { 0, 2,−4, 2, 1,−4, 0, 2,−2, 0,1,−3,−2, 2,_M,−1, 1, 0, 1, 0, 0,−2,−4, 0,−2, 1}, /* O */{_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M}, /* P */ { 1,−1,−3,−1,−1,−5,−1,0,−2, 0,−1,−3,−2,−1,_M, 6, 0, 0, 1, 0, 0,−1,−6, 0,−5, 0}, /* Q */ { 0,1,−5, 2, 2,−5,−1, 3,−2, 0, 1,−2,−1, 1,_M, 0, 4, 1,−1,−1, 0,−2,−5, 0,−4,3}, /* R */ {−2, 0,−4,−1,−1,−4,−3, 2,−2, 0, 3,−3, 0, 0,_M, 0, 1, 6,0,−1, 0,−2, 2, 0,−4, 0}, /* S */ { 1, 0, 0, 0, 0,−3, 1,−1,−1, 0,0,−3,−2, 1,_M, 1,−1, 0, 2, 1, 0,−1,−2, 0,−3, 0}, /* T */ { 1, 0,−2, 0,0,−3, 0,−1, 0, 0, 0,−1,−1, 0,_M, 0,−1,−1, 1, 3, 0, 0,−5, 0,−3, 0}, /* U*/ { 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0}, /* V */ { 0,−2,−2,−2,−2,−1,−1,−2, 4, 0,−2, 2,2,−2,_M,−1,−2,−2,−1, 0, 0, 4,−6, 0,−2,−2}, /* W */ {−6,−5,−8,−7,−7,0,−7,−3,−5, 0,−3,−2,−4,−4,_M,−6,−5, 2,−2,−5, 0,−6,17, 0, 0,−6}, /* X */{ 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0}, /* Y */ {−3,−3, 0,−4,−4, 7,−5, 0,−1,0,−4,−1,−2,−2,_M,−5,−4,−4,−3,−3, 0,−2, 0, 0,10,−4}, /* Z */ { 0, 1,−5,2, 3,−5, 0, 2,−2, 0, 0,−2,−1, 1,_M, 0, 3, 0, 0, 0, 0,−2,−6, 0,−4, 4} };/*  */ #include <stdio.h> #include <ctype.h> #define MAXJMP 16 /* maxjumps in a diag */ #define MAXGAP 24 /* don't continue to penalize gapslarger than this */ #define JMPS 1024 /* max jmps in an path */ #defineMX 4 /* save if there's at least MX−1 bases since last jmp */ #defineDMAT 3 /* value of matching bases */ #define DMIS 0 /* penalty formismatched bases */ #define DINS0 8 /* penalty for a gap */ #defineDINS1 1 /* penalty per base */ #define PINS0 8 /* penalty for a gap */#define PINS1 4 /* penalty per residue */ struct jmp { short n[MAXJMP];/* size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no.of jmp in seq x */ }; /* limits seq to 2{circumflex over ( )}16 −1 */struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs( ) */ char *prog; /* prog name for errmsgs */ char *seqx[2]; /* seqs: getseqs( ) */ int dmax; /* best diag:nw( ) */ int dmax0; /* final diag */ int dna; /* set if dna: main( ) */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw( ) */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc( ), *malloc( ), *index( ), *strcpy( ); char*getseq( ), *g_calloc( ); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  *  where file1 and file2 are two dna or twoprotein sequences.  *  The sequences can be in upper- or lower-case anmay contain ambiguity  *  Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  *  Max file length is 65535 (limited by unsigned short x in thejmp struct)  *  A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  *  Output is in the file “align.out”  *  * Theprogram may create a tmp file in /tmp to hold info about traceback.  *Original version developed under BSD 4.3 on a vax 8650  */ #include“nw.h” #include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1<<(‘D’-‘A’))|(1<<(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0xFFFFFFF, 1<<10, 1<<11, 1<<12, 1<<13, 1<<14, 1<<15, 1<<16,1<<17, 1<<18, 1<<19, 1<<20, 1<<21, 1<<22, 1<<23, 1<<24,1<<25|(1<<(‘E’-‘A’))|(1<<(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[ ]; { prog = av[0]; if (ac != 3) { fprintf(stderr,“usage: %s file1file2\n”, prog); fprintf(stderr,“where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr,“The sequences can be inupper- or lower-case\n”); fprintf(stderr,“Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr,“Output is in the file\”align.out\“\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw( ); /* fill in the matrix, getthe possible jmps */ readjmps( ); /* get the actual jmps */ print( ); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main( )  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw( ) nw { char *px, *py; /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PINS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy = 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0] − ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx =0; } ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis += (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if (col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] = −ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy); if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps && xx< len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) { smax= col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; } (void)free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print( ) -- only routinevisible outside this module  *  * static:  * getmat( ) -- trace backbest path, count matches: print( )  * pr_align( ) -- print alignment ofdescribed in array p[ ]: print( )  * dumpblock( ) -- dump a block oflines with numbers, stars: pr_align( )  * nums( ) -- put out a numberline: dumpblock( )  * putline( ) -- put out a line (name, [num], seq,[num]): dumpblock( )  * stars( ) - -put a line of stars: dumpblock( )  *stripname( ) -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC 3 #define P_LINE 256 /* maximum output line */#define P_SPC 3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print( ) print { int lx, ly, firstgap, lastgap; /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr,“%s: can't write %s\n”,prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s (length =%d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s (length =%d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1; firstgap =lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */ pp[0].spc =firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if (dmax > len1 − 1){ /* leading gap in y */ pp[1].spc = firstgap = dmax − (len1 − 1); lx −=pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gap in x */ lastgap =len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 > len0 − 1) { /*trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −= lastgap; }getmat(lx, ly, firstgap, lastgap); pr_align( ); } /*  * trace back thebest path, count matches  */ static getmat(lx, ly, firstgap, lastgap)getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap, lastgap;/* leading trailing overlap */ { int nm, i0, i1, siz0, siz1; charoutx[32]; double pct; register n0, n1; register char *p0, *p1; /* gettotal matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (n1++ == pp[1].x[i1]) siz1 = pp[1].n[i1++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “ (%d %s%s)”, ngapx, (dna)?“base”:“residue”, (ngapx == 1)? “”:“s”); fprintf(fx,“%s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “ (%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy ==1)? “”:“s”); fprintf(fx,“%s”, outx); } if (dna) fprintf(fx, “\n<score:%d (match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars( ) */ /*  * print alignment of described in struct path pp[ ] */ static pr_align( ) pr_align { int nn; /* char count */ int more;register i; for (i = 0, lmax = 0; i < 2; i++) { nn =stripname(namex[i]); if (nn > lmax) lmax = nn; nc[i] = 1; ni[i] = 1;siz[i] = ij[i] = 0; ps[i] = seqx[i]; po[i] = out[i]; } for (nn = nm = 0,more = 1; more; ) { ...pr_align for (i = more = 0; i < 2; i++) { /*  *do we have more of this sequence?  */ if (!*ps[i]) continue; more++; if(pp[i].spc) { /* leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if(siz[i]) { /* in a gap */ *po[i]++ = ‘-’; siz[i]−−; } else { /* we'reputting a seq element  */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] = pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock( ); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align( )  */ static dumpblock( ) dumpblock { register i; for(i = 0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx);for (i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) !=‘ ’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars( );putline(i); if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1)nums(i); } } } /*  * put out a number line: dumpblock( )  */ staticnums(ix) nums int ix; /* index in out[ ] holding seq line */ { charnline[P_LINE]; register i, j; register char *pn, *px, *py; for (pn =nline, i = 0; i < lmax+P_SPC; i++, pn++) *pn = ‘ ’; for (i = nc[ix], py= out[ix]; *py; py++, pn++) { if (*py == ‘ ’ || *py == ‘-’) *pn = ‘ ’;else { if (i%10 == 0 || (i == 1 && nc[ix] != 1)) { j = (i < 0)? −i : i;for (px = pn; j; j /= 10, px−−) *px = j%10 + ‘0’; if (i < 0) *px = ‘-’;} else *pn = ‘ ’; i++; } } *pn = ‘\0’; nc[ix] = i; for (pn = nline; *pn;pn++) (void) putc(*pn, fx); (void) putc(‘\n’, fx); } /*  * put out aline (name, [num], seq, [num]): dumpblock( )  */ static putline(ix)putline int ix; { ...putline int i; register char *px; for (px =namex[ix], i = 0; *px && *px != ‘:’; px++, i++) (void) putc(*px, fx);for (; i < lmax+P_SPC; i++) (void) putc(‘ ’, fx); /* these count from 1: * ni[ ] is current element (from 1)  * nc[ ] is number at start ofcurrent line  */ for (px = out[ix]; *px; px++) (void) putc(*px&0x7F,fx); (void) putc(‘\n’, fx); } /*  * put a line of stars (seqs always inout[0], out[1]): dumpblock( )  */ static stars( ) stars { int i;register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0] == ‘ ’ &&*(po[0]) == ‘ ’) ||  !*out[1] || (*out[1] == ‘ ’ && *(po[1]) == ‘ ’))return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for (p0 =out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0−‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align( )  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup( ) --cleanup any tmp file  * getseq( ) -- read in seq, set dna, len, maxlen * g_calloc( ) -- calloc( ) with error checkin  * readjmps( ) -- get thegood jmps, from tmp file if necessary  * writejmps( ) -- write a filledarray of jmps to a tmp file: nw( )  */ #include “nw.h” #include<sys/file.h> char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */FILE *fj; int cleanup( ); /* cleanup tmp file */ long lseek( ); /*  *remove any tmp file if we blow  */ cleanup(i) cleanup int i; { if (fj)(void) unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna,len, maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upperor lower case  */ char * getseq(file, len) getseq char *file; /* filename */ int *len; /* seq len */ { char line[1024], *pseq; register char*px, *py; int natgc, tlen; FILE *fp; if ((fp = fopen(file,“r”)) == 0) {fprintf(stderr,“%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr,“%s: malloc( ) failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”,*(py−1))) natgc++; } } *py++ = ‘\0’; *py= ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc( ); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc( ) failed %s (n=%d, sz=%d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[ ] or tmp file, set pp[ ], reset dmax: main( )  */ readjmps( )readjmps { int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) {(void) fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) {fprintf(stderr, “%s: can't open( ) %s\n”, prog, jname); cleanup(1); } }for (i = i0 = i1 = 0, dmax0 = dmax, xx = len0; ; i++) { while (1) { for(j = dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmpsif (j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset,0); (void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[i1] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[i1] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; i1++; } else if (siz > 0) { /* gapin first seq */ pp[0].n[i0] = siz; pp[0].x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw( )  */writejmps(ix) writejmps int ix; { char *mktemp( ); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp( ) %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj);

The term “vector,” as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA loop into which additional DNA segments maybe ligated. Another type of vector is a phage vector. Another type ofvector is a viral vector, wherein additional DNA segments may be ligatedinto the viral genome. Certain vectors are capable of autonomousreplication in a host cell into which they are introduced (e.g.,bacterial vectors having a bacterial origin of replication and episomalmammalian vectors). Other vectors (e.g., non-episomal mammalian vectors)can be integrated into the genome of a host cell upon introduction intothe host cell, and thereby are replicated along with the host genome.Moreover, certain vectors are capable of directing the expression ofgenes to which they are operatively linked. Such vectors are referred toherein as “recombinant expression vectors” (or simply, “recombinantvectors”). In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” may be used interchangeably as theplasmid is the most commonly used form of vector.

“Polynucleotide,” or “nucleic acid,” as used interchangeably herein,refer to polymers of nucleotides of any length, and include DNA and RNA.The nucleotides can be deoxyribonucleotides, ribonucleotides, modifiednucleotides or bases, and/or their analogs, or any substrate that can beincorporated into a polymer by DNA or RNA polymerase, or by a syntheticreaction. A polynucleotide may comprise modified nucleotides, such asmethylated nucleotides and their analogs. If present, modification tothe nucleotide structure may be imparted before or after assembly of thepolymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter synthesis, such as by conjugation with a label. Other types ofmodifications include, for example, “caps”, substitution of one or moreof the naturally occurring nucleotides with an analog, internucleotidemodifications such as, for example, those with uncharged linkages (e.g.,methyl phosphonates, phosphotriesters, phosphoamidates, carbamates,etc.) and with charged linkages (e.g., phosphorothioates,phosphorodithioates, etc.), those containing pendant moieties, such as,for example, proteins (e.g., nucleases, toxins, antibodies, signalpeptides, poly-L-lysine, etc.), those with intercalators (e.g.,acridine, psoralen, etc.), those containing chelators (e.g., metals,radioactive metals, boron, oxidative metals, etc.), those containingalkylators, those with modified linkages (e.g., alpha anomeric nucleicacids, etc.), as well as unmodified forms of the polynucleotide(s).Further, any of the hydroxyl groups ordinarily present in the sugars maybe replaced, for example, by phosphonate groups, phosphate groups,protected by standard protecting groups, or activated to prepareadditional linkages to additional nucleotides, or may be conjugated tosolid or semi-solid supports. The 5′ and 3′ terminal OH can bephosphorylated or substituted with amines or organic capping groupmoieties of from 1 to 20 carbon atoms. Other hydroxyls may also bederivatized to standard protecting groups. Polynucleotides can alsocontain analogous forms of ribose or deoxyribose sugars that aregenerally known in the art, including, for example, 2′-O-methyl-,2′-O-allyl, 2′-fluoro- or 2′-azido-ribose, carbocyclic sugar analogs,alpha-anomeric sugars, epimeric sugars such as arabinose, xyloses orlyxoses, pyranose sugars, furanose sugars, sedoheptuloses, acyclicanalogs and abasic nucleoside analogs such as methyl riboside. One ormore phosphodiester linkages may be replaced by alternative linkinggroups. These alternative linking groups include, but are not limitedto, embodiments wherein phosphate is replaced by P(O)S(“thioate”), P(S)S(“dithioate”), “(O)NR₂ (“amidate”), P(O)R, P(O)OR′, CO or CH2(“formacetal”), in which each R or R′ is independently H or substitutedor unsubstituted alkyl (1-20 C.) optionally containing an ether (—O—)linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or araldyl. Not alllinkages in a polynucleotide need be identical. The precedingdescription applies to all polynucleotides referred to herein, includingRNA and DNA.

“Oligonucleotide,” as used herein, generally refers to short, generallysingle stranded, generally synthetic polynucleotides that are generally,but not necessarily, less than about 200 nucleotides in length. Theterms “oligonucleotide” and “polynucleotide” are not mutually exclusive.The description above for polynucleotides is equally and fullyapplicable to oligonucleotides.

The term “hepatocyte growth factor” or “HGF”, as used herein, refers,unless specifically or contextually indicated otherwise, to any nativeor variant (whether native or synthetic) HGF polypeptide that is capableof activating the HGF/c-met signaling pathway under conditions thatpermit such process to occur. The term “wild type HGF” generally refersto a polypeptide comprising the amino acid sequence of a naturallyoccurring HGF protein. The term “wild type HGF sequence” generallyrefers to an amino acid sequence found in a naturally occurring HGF.

The terms “antibody” and “immunoglobulin” are used interchangeably inthe broadest sense and include monoclonal antibodies (e.g., full lengthor intact monoclonal antibodies), polyclonal antibodies, multivalentantibodies, multispecific antibodies (e.g., bispecific antibodies solong as they exhibit the desired biological activity) and may alsoinclude certain antibody fragments (as described in greater detailherein). An antibody can be human, humanized and/or affinity matured.

“Antibody fragments” comprise only a portion of an intact antibody,wherein the portion preferably retains at least one, preferably most orall, of the functions normally associated with that portion when presentin an intact antibody. In one embodiment, an antibody fragment comprisesan antigen binding site of the intact antibody and thus retains theability to bind antigen. In another embodiment, an antibody fragment,for example one that comprises the Fc region, retains at least one ofthe biological functions normally associated with the Fc region whenpresent in an intact antibody, such as FcRn binding, antibody half lifemodulation, ADCC function and complement binding. In one embodiment, anantibody fragment is a monovalent antibody that has an in vivo half lifesubstantially similar to an intact antibody. For example, such anantibody fragment may comprise one antigen binding arm linked to an Fcsequence capable of conferring in vivo stability to the fragment. In oneembodiment, an antibody of the invention is a one-armed antibody asdescribed in WO2005/063816. In one embodiment, the one-armed antibodycomprises Fc mutations constituting “knobs” and “holes” as described inWO2005/063816. For example, a hole mutation can be one or more of T366A,L368A and/or Y407V in an Fc polypeptide, and a knob mutation can beT366W.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigen. Furthermore, in contrast to polyclonalantibody preparations that typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody is directed against a single determinant on the antigen.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison etal., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or nonhuman primate having the desired specificity,affinity, and capacity. In some instances, framework region (FR)residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the followingreview articles and references cited therein: Vaswani and Hamilton, Ann.Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc.Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech.5:428-433 (1994).

An “antigen” is a predetermined antigen to which an antibody canselectively bind. The target antigen may be polypeptide, carbohydrate,nucleic acid, lipid, hapten or other naturally occurring or syntheticcompound. Preferably, the target antigen is a polypeptide. An “acceptorhuman framework” for the purposes herein is a framework comprising theamino acid sequence of a VL or VH framework derived from a humanimmunoglobulin framework, or from a human consensus framework. Anacceptor human framework “derived from” a human immunoglobulin frameworkor human consensus framework may comprise the same amino acid sequencethereof, or may contain pre-existing amino acid sequence changes. Wherepre-existing amino acid changes are present, preferably no more than 5and preferably 4 or less, or 3 or less, pre-existing amino acid changesare present. Where pre-existing amino acid changes are present in a VH,preferably those changes are only at three, two or one of positions 71H,73H and 78H; for instance, the amino acid residues at those positionsmay be 71A, 73T and/or 78A. In one embodiment, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residue in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al. In one embodiment, for the VL, the subgroup is subgroupkappa I as in Kabat et al. In one embodiment, for the VH, the subgroupis subgroup III as in Kabat et al.

A “VH subgroup III consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable heavy subgroup III ofKabat et al. In one embodiment, the VH subgroup III consensus frameworkamino acid sequence comprises at least a portion or all of each of thefollowing sequences:

(SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 43)-H1-WVRQAPGKGLEWV (SEQ ID NO: 44) -H2-RFTISRDNSKNTLYLQMNSLRAEDTAVYYC(SEQ ID NO: 45) -H3-WGQGTLVTVSS.

A “VL subgroup I consensus framework” comprises the consensus sequenceobtained from the amino acid sequences in variable light kappa subgroupI of Kabat et al. In one embodiment, the VH subgroup I consensusframework amino acid sequence comprises at least a portion or all ofeach of the following sequences:

(SEQ ID NO: 46) DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 47)-L1-WYQQKPGKAPKLLIY (SEQ ID NO: 48) -L2-GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC(SEQ ID NO: 49) -L3-FGQGTKVEIK.

An “unmodified human framework” is a human framework which has the sameamino acid sequence as the acceptor human framework, e.g. lacking humanto non-human amino acid substitution(s) in the acceptor human framework.

An “altered hypervariable region” for the purposes herein is ahypervariable region comprising one or more (e.g. one to about 16) aminoacid substitution(s) therein.

An “un-modified hypervariable region” for the purposes herein is ahypervariable region having the same amino acid sequence as a non-humanantibody from which it was derived, i.e. one which lacks one or moreamino acid substitutions therein.

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH(H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM hypervariable regions representa compromise between the Kabat CDRs and Chothia structural loops, andare used by Oxford Molecular's AbM antibody modeling software. The“contact” hypervariable regions are based on an analysis of theavailable complex crystal structures. The residues from each of thesehypervariable regions are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

Hypervariable regions may comprise “extended hypervariable regions” asfollows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2) and 89-97 (L3) in theVL and 26-35 (H1), 50-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3)in the VH. The variable domain residues are numbered according to Kabatet al., supra for each of these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to a parent antibody which doesnot possess those alteration(s). Preferred affinity matured antibodieswill have nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced by procedures known in the art.Marks et al. Bio/Technology 10:779-783 (1992) describes affinitymaturation by VH and VL domain shuffling. Random mutagenesis of CDRand/or framework residues is described by: Barbas et al. Proc Nat. Acad.Sci, USA 91:3809-3813 (1994); Schier et al. Gene 169:147-155 (1995);Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J.Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol.226:889-896 (1992).

A “blocking” antibody or an “antagonist” antibody is one which inhibitsor reduces biological activity of the antigen it bind. Preferredblocking antibodies or antagonist antibodies substantially or completelyinhibit the biological activity of the antigen.

An “agonist antibody”, as used herein, is an antibody which mimics atleast one of the functional activities of a polypeptide of interest.

A “disorder” is any condition that would benefit from treatment with asubstance/molecule or method of the invention. This includes chronic andacute disorders or diseases including those pathological conditionswhich predispose the mammal to the disorder in question. Non-limitingexamples of disorders to be treated herein include malignant and benigntumors; non-leukemias and lymphoid malignancies; neuronal, glial,astrocytal, hypothalamic and other glandular, macrophagal, epithelial,stromal and blastocoelic disorders; and inflammatory, immunologic andother angiogenesis-related disorders.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth/proliferation. Examples of cancer include butare not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia. More particular examples of such cancers include squamous cellcancer, small-cell lung cancer, non-small cell lung cancer,adenocarcinoma of the lung, squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastrointestinal cancer,pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, livercancer, bladder cancer, hepatoma, breast cancer, colon cancer,colorectal cancer, endometrial or uterine carcinoma, salivary glandcarcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer,thyroid cancer, hepatic carcinoma and various types of head and neckcancer.

Dysregulation of angiogenesis can lead to many disorders that can betreated by compositions and methods of the invention. These disordersinclude both non-neoplastic and neoplastic conditions. Neoplasticsinclude but are not limited those described above. Non-neoplasticdisorders include but are not limited to undesired or aberranthypertrophy, arthritis, rheumatoid arthritis (RA), psoriasis, psoriaticplaques, sarcoidosis, atherosclerosis, atherosclerotic plaques, diabeticand other proliferative retinopathies including retinopathy ofprematurity, retrolental fibroplasia, neovascular glaucoma, age-relatedmacular degeneration, diabetic macular edema, cornealneovascularization, corneal graft neovascularization, corneal graftrejection, retinal/choroidal neovascularization, neovascularization ofthe angle (rubeosis), ocular neovascular disease, vascular restenosis,arteriovenous malformations (AVM), meningioma, hemangioma, angiofibroma,thyroid hyperplasias (including Grave's disease), corneal and othertissue transplantation, chronic inflammation, lung inflammation, acutelung injury/ARDS, sepsis, primary pulmonary hypertension, malignantpulmonary effusions, cerebral edema (e.g., associated with acutestroke/closed head injury/trauma), synovial inflammation, pannusformation in RA, myositis ossificans, hypertropic bone formation,osteoarthritis (OA), refractory ascites, polycystic ovarian disease,endometriosis, 3rd spacing of fluid diseases (pancreatitis, compartmentsyndrome, burns, bowel disease), uterine fibroids, premature labor,chronic inflammation such as IBD (Crohn's disease and ulcerativecolitis), renal allograft rejection, inflammatory bowel disease,nephrotic syndrome, undesired or aberrant tissue mass growth(non-cancer), hemophilic joints, hypertrophic scars, inhibition of hairgrowth, Osler-Weber syndrome, pyogenic granuloma retrolentalfibroplasias, scleroderma, trachoma, vascular adhesions, synovitis,dermatitis, preeclampsia, ascites, pericardial effusion (such as thatassociated with pericarditis), and pleural effusion.

As used herein, “treatment” refers to clinical intervention in anattempt to alter the natural course of the individual or cell beingtreated, and can be performed either for prophylaxis or during thecourse of clinical pathology. Desirable effects of treatment includepreventing occurrence or recurrence of disease, alleviation of symptoms,diminishment of any direct or indirect pathological consequences of thedisease, preventing metastasis, decreasing the rate of diseaseprogression, amelioration or palliation of the disease state, andremission or improved prognosis. In some embodiments, antibodies of theinvention are used to delay development of a disease or disorder.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A “therapeutically effective amount” of a substance/molecule of theinvention, agonist or antagonist may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the substance/molecule, agonist or antagonist to elicit adesired response in the individual. A therapeutically effective amountis also one in which any toxic or detrimental effects of thesubstance/molecule, agonist or antagonist are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typicallybut not necessarily, since a prophylactic dose is used in subjects priorto or at an earlier stage of disease, the prophylactically effectiveamount will be less than the therapeutically effective amount.

The term “cytotoxic agent” as used herein refers to a substance thatinhibits or prevents the function of cells and/or causes destruction ofcells. The term is intended to include radioactive isotopes (e.g.,At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³² and radioactiveisotopes of Lu), chemotherapeutic agents e.g. methotrexate, adriamicin,vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin,melphalan, mitomycin C, chlorambucil, daunorubicin or otherintercalating agents, enzymes and fragments thereof such as nucleolyticenzymes, antibiotics, and toxins such as small molecule toxins orenzymatically active toxins of bacterial, fungal, plant or animalorigin, including fragments and/or variants thereof, and the variousantitumor or anticancer agents disclosed below. Other cytotoxic agentsare described below. A tumoricidal agent causes destruction of tumorcells.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includealkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,trietylenephosphoramide, triethiylenethiophosphoramide andtrimethylolomelamine; acetogenins (especially bullatacin andbullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®);beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin(including the synthetic analogue topotecan (HYCAMTIN®), CPT-11(irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including itsadozelesin, carzelesin and bizelesin synthetic analogues);podophyllotoxin; podophyllinic acid; teniposide; cryptophycins(particularly cryptophycin 1 and cryptophycin 8); dolastatin;duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1);eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogenmustards such as chlorambucil, chlornaphazine, cholophosphamide,estramustine, ifosfamide, mechlorethamine, mechlorethamine oxidehydrochloride, melphalan, novembichin, phenesterine, prednimustine,trofosfamide, uracil mustard; nitrosureas such as carmustine,chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine;antibiotics such as the enediyne antibiotics (e.g., calicheamicin,especially calicheamicin gamma1I and calicheamicin omegaI1 (see, e.g.,Agnew, Chem. Intl. Ed. Engl., 33: 183-186 (1994)); dynemicin, includingdynemicin A; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;2-ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS NaturalProducts, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium;tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine;trichothecenes (especially T-2 toxin, verracurin A, roridin A andanguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); thiotepa; taxoids, e.g., TAXOL® paclitaxel(Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil;gemcitabine (GEMZAR®); 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine(VELBAN®); platinum; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine (ONCOVIN®); oxaliplatin; leucovovin; vinorelbine(NAVELBINE®); novantrone; edatrexate; daunomycin; aminopterin;ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine(DMFO); retinoids such as retinoic acid; capecitabine (XELODA®);pharmaceutically acceptable salts, acids or derivatives of any of theabove; as well as combinations of two or more of the above such as CHOP,an abbreviation for a combined therapy of cyclophosphamide, doxorubicin,vincristine, and prednisolone, and FOLFOX, an abbreviation for atreatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU andleucovovin.

Also included in this definition are anti-hormonal agents that act toregulate, reduce, block, or inhibit the effects of hormones that canpromote the growth of cancer, and are often in the form of systemic, orwhole-body treatment. They may be hormones themselves. Examples includeanti-estrogens and selective estrogen receptor modulators (SERMs),including, for example, tamoxifen (including NOLVADEX® tamoxifen),EVISTA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY117018, onapristone, and FARESTON® toremifene;anti-progesterones; estrogen receptor down-regulators (ERDs); agentsthat function to suppress or shut down the ovaries, for example,leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON®and ELIGARD® leuprolide acetate, goserelin acetate, buserelin acetateand tripterelin; other anti-androgens such as flutamide, nilutamide andbicalutamide; and aromatase inhibitors that inhibit the enzymearomatase, which regulates estrogen production in the adrenal glands,such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE®megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole,RIVISOR® vorozole, FEMARA® letrozole, and ARIMIDEX® anastrozole. Inaddition, such definition of chemotherapeutic agents includesbisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),DIDROCAL® etidronate, NE-58095, ZOMETA® zoledronic acid/zoledronate,FOSAMAX® alendronate, AREDIA® pamidronate, SKELID® tiludronate, orACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolanenucleoside cytosine analog); antisense oligonucleotides, particularlythose that inhibit expression of genes in signaling pathways implicatedin abherant cell proliferation, such as, for example, PKC-alpha, Raf,H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such asTHERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN®vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; LURTOTECAN®topoisomerase 1 inhibitor; ABARELIX® rmRH; lapatinib ditosylate (anErbB-2 and EGFR dual tyrosine kinase small-molecule inhibitor also knownas GW572016); and pharmaceutically acceptable salts, acids orderivatives of any of the above.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell whose growth is dependentupon HGF/c-met activation either in vitro or in vivo. Thus, the growthinhibitory agent may be one which significantly reduces the percentageof HGF/c-met-dependent cells in S phase. Examples of growth inhibitoryagents include agents that block cell cycle progression (at a placeother than S phase), such as agents that induce G1 arrest and M-phasearrest. Classical M-phase blockers include the vincas (vincristine andvinblastine), taxanes, and topoisomerase II inhibitors such asdoxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Thoseagents that arrest G1 also spill over into S-phase arrest, for example,DNA alkylating agents such as tamoxifen, prednisone, dacarbazine,mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C.Further information can be found in The Molecular Basis of Cancer,Mendelsohn and Israel, eds., Chapter 1, entitled “Cell cycle regulation,oncogenes, and antineoplastic drugs” by Murakami et al. (WB Saunders:Philadelphia, 1995), especially p. 13. The taxanes (paclitaxel anddocetaxel) are anticancer drugs both derived from the yew tree.Docetaxel (TAXOTERE®, Rhone-Poulenc Rorer), derived from the Europeanyew, is a semisynthetic analogue of paclitaxel (TAXOL®, Bristol-MyersSquibb). Paclitaxel and docetaxel promote the assembly of microtubulesfrom tubulin dimers and stabilize microtubules by preventingdepolymerization, which results in the inhibition of mitosis in cells.

“Doxorubicin” is an anthracycline antibiotic. The full chemical name ofdoxorubicin is(8S-cis)-10-[(3-amino-2,3,6-trideoxy-α-L-lyxo-hexapyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-5,12-naphthacenedione.

Generating Variant Antibodies Exhibiting Reduced or Absence of HAMAResponse

Reduction or elimination of a HAMA response is a significant aspect ofclinical development of suitable therapeutic agents. See, e.g.,Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al.,Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985),135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller etal., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352;Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res.(1990), 50:1495. As described herein, the invention provides antibodiesthat are humanized such that HAMA response is reduced or eliminated.Variants of these antibodies can further be obtained using routinemethods known in the art, some of which are further described below.

For example, an amino acid sequence from an antibody as described hereincan serve as a starting (parent) sequence for diversification of theframework and/or hypervariable sequence(s). A selected frameworksequence to which a starting hypervariable sequence is linked isreferred to herein as an acceptor human framework. While the acceptorhuman frameworks may be from, or derived from, a human immunoglobulin(the VL and/or VH regions thereof), preferably the acceptor humanframeworks are from, or derived from, a human consensus frameworksequence as such frameworks have been demonstrated to have minimal, orno, immunogenicity in human patients.

Where the acceptor is derived from a human immunoglobulin, one mayoptionally select a human framework sequence that is selected based onits homology to the donor framework sequence by aligning the donorframework sequence with various human framework sequences in acollection of human framework sequences, and select the most homologousframework sequence as the acceptor.

In one embodiment, human consensus frameworks herein are from, orderived from, VH subgroup III and/or VL kappa subgroup I consensusframework sequences.

Thus, the VH acceptor human framework may comprise one, two, three orall of the following framework sequences:

FR1 comprising (SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAAS, FR2 comprising(SEQ ID NO: 43) WVRQAPGKGLEWV, FR3 comprising FR3 comprises(SEQ ID NO: 50) RFTISX1DX2SKNTX3YLQMNSLRAEDTAVYYC,wherein X1 is A or R, X2 is T or N, and X3 is A or L, FR4 comprising(SEQ ID NO: 45) WGQGTLVTVSS.Examples of VH consensus frameworks include:human VH subgroup I consensus framework minus Kabat CDRs (SEQ ID NO:19);human VH subgroup I consensus framework minus extended hypervariableregions (SEQ ID NOs:20-22);human VH subgroup II consensus framework minus Kabat CDRs (SEQ IDNO:23);human VH subgroup II consensus framework minus extended hypervariableregions (SEQ ID NOs:24-26);human VH subgroup III consensus framework minus Kabat CDRs (SEQ IDNO:27);human VH subgroup III consensus framework minus extended hypervariableregions (SEQ ID NO:28-30);human VH acceptor framework minus Kabat CDRs (SEQ ID NO:31);human VH acceptor framework minus extended hypervariable regions (SEQ IDNOs:32-33);human VH acceptor 2 framework minus Kabat CDRs (SEQ ID NO:34); orhuman VH acceptor 2 framework minus extended hypervariable regions (SEQID NOs:35-37).

In one embodiment, the VH acceptor human framework comprises one, two,three or all of the following framework sequences:

FR1 comprising (SEQ ID NO: 42) EVQLVESGGGLVQPGGSLRLSCAAS, FR2 comprising(SEQ ID NO: 43) WVRQAPGKGLEWV, FR3 comprising (SEQ ID NO: 51)RFTISADTSKNTAYLQMNSLRAEDTAVYYC, (SEQ ID NO: 52)RFTISADTSKNTAYLQMNSLRAEDTAVYYCA, (SEQ ID NO: 53)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR, (SEQ ID NO: 54)RFTISADTSKNTAYLQMNSLRAEDTAVYYCS, or (SEQ ID NO: 55)RFTISADTSKNTAYLQMNSLRAEDTAVYYCSR FR4 comprising (SEQ ID NO: 45)WGQGTLVTVSS.

The VL acceptor human framework may comprise one, two, three or all ofthe following framework sequences:

FR1 comprising (SEQ ID NO: 46) DIQMTQSPSSLSASVGDRVTITC, FR2 comprising(SEQ ID NO: 47) WYQQKPGKAPKLLIY, FR3 comprising (SEQ ID NO: 48)GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC, FR4 comprising (SEQ ID NO: 49)FGQGTKVEIK.Examples of VL consensus frameworks include:human VL kappa subgroup I consensus framework (SEQ ID NO:38);human VL kappa subgroup II consensus framework (SEQ ID NO:39);human VL kappa subgroup III consensus framework (SEQ ID NO:40); orhuman VL kappa subgroup IV consensus framework (SEQ ID NO:41)

While the acceptor may be identical in sequence to the human frameworksequence selected, whether that be from a human immunoglobulin or ahuman consensus framework, the present invention contemplates that theacceptor sequence may comprise pre-existing amino acid substitutionsrelative to the human immunoglobulin sequence or human consensusframework sequence. These pre-existing substitutions are preferablyminimal; usually four, three, two or one amino acid differences onlyrelative to the human immunoglobulin sequence or consensus frameworksequence.

Hypervariable region residues of the non-human antibody are incorporatedinto the VL and/or VH acceptor human frameworks. For example, one mayincorporate residues corresponding to the Kabat CDR residues, theChothia hypervariable loop residues, the Abm residues, and/or contactresidues. Optionally, the extended hypervariable region residues asfollows are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35(H1), 50-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3).

While “incorporation” of hypervariable region residues is discussedherein, it will be appreciated that this can be achieved in variousways, for example, nucleic acid encoding the desired amino acid sequencecan be generated by mutating nucleic acid encoding the mouse variabledomain sequence so that the framework residues thereof are changed toacceptor human framework residues, or by mutating nucleic acid encodingthe human variable domain sequence so that the hypervariable domainresidues are changed to non-human residues, or by synthesizing nucleicacid encoding the desired sequence, etc.

In the examples herein, hypervariable region-grafted variants weregenerated by Kunkel mutagenesis of nucleic acid encoding the humanacceptor sequences, using a separate oligonucleotide for eachhypervariable region. Kunkel et al., Methods Enzymol. 154:367-382(1987). Appropriate changes can be introduced within the frameworkand/or hypervariable region, using routine techniques, to correct andre-establish proper hypervariable region-antigen interactions.

Phage(mid) display (also referred to herein as phage display in somecontexts) can be used as a convenient and fast method for generating andscreening many different potential variant antibodies in a librarygenerated by sequence randomization. However, other methods for makingand screening altered antibodies are available to the skilled person.

Phage(mid) display technology has provided a powerful tool forgenerating and selecting novel proteins which bind to a ligand, such asan antigen. Using the techniques of phage(mid) display allows thegeneration of large libraries of protein variants which can be rapidlysorted for those sequences that bind to a target molecule with highaffinity. Nucleic acids encoding variant polypeptides are generallyfused to a nucleic acid sequence encoding a viral coat protein, such asthe gene III protein or the gene VIII protein. Monovalent phagemiddisplay systems where the nucleic acid sequence encoding the protein orpolypeptide is fused to a nucleic acid sequence encoding a portion ofthe gene III protein have been developed. (Bass, S., Proteins, 8:309(1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology,3:205 (1991)). In a monovalent phagemid display system, the gene fusionis expressed at low levels and wild type gene III proteins are alsoexpressed so that infectivity of the particles is retained. Methods ofgenerating peptide libraries and screening those libraries have beendisclosed in many patents (e.g. U.S. Pat. No. 5,723,286, U.S. Pat. No.5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S.Pat. No. 5,498,530).

Libraries of antibodies or antigen binding polypeptides have beenprepared in a number of ways including by altering a single gene byinserting random DNA sequences or by cloning a family of related genes.Methods for displaying antibodies or antigen binding fragments usingphage(mid) display have been described in U.S. Pat. Nos. 5,750,373,5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The library is then screened for expression of antibodies or antigenbinding proteins with the desired characteristics.

Methods of substituting an amino acid of choice into a template nucleicacid are well established in the art, some of which are describedherein. For example, hypervariable region residues can be substitutedusing the Kunkel method. See, e.g., Kunkel et al., Methods Enzymol.154:367-382 (1987).

The sequence of oligonucleotides includes one or more of the designedcodon sets for the hypervariable region residues to be altered. A codonset is a set of different nucleotide triplet sequences used to encodedesired variant amino acids. Codon sets can be represented using symbolsto designate particular nucleotides or equimolar mixtures of nucleotidesas shown in below according to the IUB code.

  IUB CODES G Guanine A Adenine T Thymine C Cytosine R (A or G) Y (C orT) M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C or Gor T) V (A or C or G) D (A or G or T) N (A or C or G or T)

For example, in the codon set DVK, D can be nucleotides A or G or T; Vcan be A or G or C; and K can be G or T. This codon set can present 18different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr,Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

Oligonucleotide or primer sets can be synthesized using standardmethods. A set of oligonucleotides can be synthesized, for example, bysolid phase synthesis, containing sequences that represent all possiblecombinations of nucleotide triplets provided by the codon set and thatwill encode the desired group of amino acids. Synthesis ofoligonucleotides with selected nucleotide “degeneracy” at certainpositions is well known in that art. Such sets of nucleotides havingcertain codon sets can be synthesized using commercial nucleic acidsynthesizers (available from, for example, Applied Biosystems, FosterCity, Calif.), or can be obtained commercially (for example, from LifeTechnologies, Rockville, Md.). Therefore, a set of oligonucleotidessynthesized having a particular codon set will typically include aplurality of oligonucleotides with different sequences, the differencesestablished by the codon set within the overall sequence.Oligonucleotides, as used according to the invention, have sequencesthat allow for hybridization to a variable domain nucleic acid templateand also can include restriction enzyme sites for cloning purposes.

In one method, nucleic acid sequences encoding variant amino acids canbe created by oligonucleotide-mediated mutagenesis. This technique iswell known in the art as described by Zoller et al. Nucleic Acids Res.10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variantamino acids are created by hybridizing an oligonucleotide set encodingthe desired codon sets to a DNA template, where the template is thesingle-stranded form of the plasmid containing a variable region nucleicacid template sequence. After hybridization, DNA polymerase is used tosynthesize an entire second complementary strand of the template thatwill thus incorporate the oligonucleotide primer, and will contain thecodon sets as provided by the oligonucleotide set.

Generally, oligonucleotides of at least 25 nucleotides in length areused. An optimal oligonucleotide will have 12 to 15 nucleotides that arecompletely complementary to the template on either side of thenucleotide(s) coding for the mutation(s). This ensures that theoligonucleotide will hybridize properly to the single-stranded DNAtemplate molecule. The oligonucleotides are readily synthesized usingtechniques known in the art such as that described by Crea et al., Proc.Nat'l. Acad. Sci. USA, 75:5765 (1978).

The DNA template is generated by those vectors that are either derivedfrom bacteriophage M13 vectors (the commercially available M13 mp18 andM13 mp19 vectors are suitable), or those vectors that contain asingle-stranded phage origin of replication as described by Viera etal., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutatedcan be inserted into one of these vectors in order to generatesingle-stranded template. Production of the single-stranded template isdescribed in sections 4.21-4.41 of Sambrook et al., above.

To alter the native DNA sequence, the oligonucleotide is hybridized tothe single stranded template under suitable hybridization conditions. ADNA polymerizing enzyme, usually T7 DNA polymerase or the Klenowfragment of DNA polymerase I, is then added to synthesize thecomplementary strand of the template using the oligonucleotide as aprimer for synthesis. A heteroduplex molecule is thus formed such thatone strand of DNA encodes the mutated form of gene 1, and the otherstrand (the original template) encodes the native, unaltered sequence ofgene 1. This heteroduplex molecule is then transformed into a suitablehost cell, usually a prokaryote such as E. coli JM101. After growing thecells, they are plated onto agarose plates and screened using theoligonucleotide primer radiolabelled with a 32-Phosphate to identify thebacterial colonies that contain the mutated DNA.

The method described immediately above may be modified such that ahomoduplex molecule is created wherein both strands of the plasmidcontain the mutation(s). The modifications are as follows: The singlestranded oligonucleotide is annealed to the single-stranded template asdescribed above. A mixture of three deoxyribonucleotides,deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), anddeoxyribothymidine (dTT), is combined with a modifiedthiodeoxyribocytosine called dCTP-(aS) (which can be obtained fromAmersham). This mixture is added to the template-oligonucleotidecomplex. Upon addition of DNA polymerase to this mixture, a strand ofDNA identical to the template except for the mutated bases is generated.In addition, this new strand of DNA will contain dCTP-(aS) instead ofdCTP, which serves to protect it from restriction endonucleasedigestion. After the template strand of the double-stranded heteroduplexis nicked with an appropriate restriction enzyme, the template strandcan be digested with ExoIII nuclease or another appropriate nucleasepast the region that contains the site(s) to be mutagenized. Thereaction is then stopped to leave a molecule that is only partiallysingle-stranded. A complete double-stranded DNA homoduplex is thenformed using DNA polymerase in the presence of all fourdeoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplexmolecule can then be transformed into a suitable host cell.

As indicated previously the sequence of the oligonucleotide set is ofsufficient length to hybridize to the template nucleic acid and mayalso, but does not necessarily, contain restriction sites. The DNAtemplate can be generated by those vectors that are either derived frombacteriophage M13 vectors or vectors that contain a single-strandedphage origin of replication as described by Viera et al. Meth. Enzymol.,153:3 (1987). Thus, the DNA that is to be mutated must be inserted intoone of these vectors in order to generate single-stranded template.Production of the single-stranded template is described in sections4.21-4.41 of Sambrook et al., supra.

According to another method, a library can be generated by providingupstream and downstream oligonucleotide sets, each set having aplurality of oligonucleotides with different sequences, the differentsequences established by the codon sets provided within the sequence ofthe oligonucleotides. The upstream and downstream oligonucleotide sets,along with a variable domain template nucleic acid sequence, can be usedin a polymerase chain reaction to generate a “library” of PCR products.The PCR products can be referred to as “nucleic acid cassettes”, as theycan be fused with other related or unrelated nucleic acid sequences, forexample, viral coat proteins and dimerization domains, using establishedmolecular biology techniques.

The sequence of the PCR primers includes one or more of the designedcodon sets for the solvent accessible and highly diverse positions in ahypervariable region. As described above, a codon set is a set ofdifferent nucleotide triplet sequences used to encode desired variantamino acids.

Antibody selectants that meet the desired criteria, as selected throughappropriate screening/selection steps can be isolated and cloned usingstandard recombinant techniques.

Vectors, Host Cells and Recombinant Methods

For recombinant production of an antibody of the invention, the nucleicacid encoding it is isolated and inserted into a replicable vector forfurther cloning (amplification of the DNA) or for expression. DNAencoding the antibody is readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The choice ofvector depends in part on the host cell to be used. Generally, preferredhost cells are of either prokaryotic or eukaryotic (generally mammalian)origin.

Generating Antibodies Using Prokaryotic Host Cells: Vector Construction

Polynucleotide sequences encoding polypeptide components of the antibodyof the invention can be obtained using standard recombinant techniques.Desired polynucleotide sequences may be isolated and sequenced fromantibody producing cells such as hybridoma cells. Alternatively,polynucleotides can be synthesized using nucleotide synthesizer or PCRtechniques. Once obtained, sequences encoding the polypeptides areinserted into a recombinant vector capable of replicating and expressingheterologous polynucleotides in prokaryotic hosts. Many vectors that areavailable and known in the art can be used for the purpose of thepresent invention. Selection of an appropriate vector will depend mainlyon the size of the nucleic acids to be inserted into the vector and theparticular host cell to be transformed with the vector. Each vectorcontains various components, depending on its function (amplification orexpression of heterologous polynucleotide, or both) and itscompatibility with the particular host cell in which it resides. Thevector components generally include, but are not limited to: an originof replication, a selection marker gene, a promoter, a ribosome bindingsite (RBS), a signal sequence, the heterologous nucleic acid insert anda transcription termination sequence.

In general, plasmid vectors containing replicon and control sequenceswhich are derived from species compatible with the host cell are used inconnection with these hosts. The vector ordinarily carries a replicationsite, as well as marking sequences which are capable of providingphenotypic selection in transformed cells. For example, E. coli istypically transformed using pBR322, a plasmid derived from an E. colispecies. pBR322 contains genes encoding ampicillin (Amp) andtetracycline (Tet) resistance and thus provides easy means foridentifying transformed cells. pBR322, its derivatives, or othermicrobial plasmids or bacteriophage may also contain, or be modified tocontain, promoters which can be used by the microbial organism forexpression of endogenous proteins. Examples of pBR322 derivatives usedfor expression of particular antibodies are described in detail inCarter et al., U.S. Pat. No. 5,648,237.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used astransforming vectors in connection with these hosts. For example,bacteriophage such as λGEM™-11 may be utilized in making a recombinantvector which can be used to transform susceptible host cells such as E.coli LE392.

The expression vector of the invention may comprise two or morepromoter-cistron pairs, encoding each of the polypeptide components. Apromoter is an untranslated regulatory sequence located upstream (5′) toa cistron that modulates its expression. Prokaryotic promoters typicallyfall into two classes, inducible and constitutive. Inducible promoter isa promoter that initiates increased levels of transcription of thecistron under its control in response to changes in the culturecondition, e.g. the presence or absence of a nutrient or a change intemperature.

A large number of promoters recognized by a variety of potential hostcells are well known. The selected promoter can be operably linked tocistron DNA encoding the light or heavy chain by removing the promoterfrom the source DNA via restriction enzyme digestion and inserting theisolated promoter sequence into the vector of the invention. Both thenative promoter sequence and many heterologous promoters may be used todirect amplification and/or expression of the target genes. In someembodiments, heterologous promoters are utilized, as they generallypermit greater transcription and higher yields of expressed target geneas compared to the native target polypeptide promoter.

Promoters suitable for use with prokaryotic hosts include the PhoApromoter, the β-galactamase and lactose promoter systems, a tryptophan(trp) promoter system and hybrid promoters such as the tac or the trcpromoter. However, other promoters that are functional in bacteria (suchas other known bacterial or phage promoters) are suitable as well. Theirnucleotide sequences have been published, thereby enabling a skilledworker operably to ligate them to cistrons encoding the target light andheavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers oradaptors to supply any required restriction sites.

In one aspect of the invention, each cistron within the recombinantvector comprises a secretion signal sequence component that directstranslocation of the expressed polypeptides across a membrane. Ingeneral, the signal sequence may be a component of the vector, or it maybe a part of the target polypeptide DNA that is inserted into thevector. The signal sequence selected for the purpose of this inventionshould be one that is recognized and processed (i.e. cleaved by a signalpeptidase) by the host cell. For prokaryotic host cells that do notrecognize and process the signal sequences native to the heterologouspolypeptides, the signal sequence is substituted by a prokaryotic signalsequence selected, for example, from the group consisting of thealkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II(STII) leaders, LamB, PhoE, Pe1B, OmpA and MBP. In one embodiment of theinvention, the signal sequences used in both cistrons of the expressionsystem are STII signal sequences or variants thereof.

In another aspect, the production of the immunoglobulins according tothe invention can occur in the cytoplasm of the host cell, and thereforedoes not require the presence of secretion signal sequences within eachcistron. In that regard, immunoglobulin light and heavy chains areexpressed, folded and assembled to form functional immunoglobulinswithin the cytoplasm. Certain host strains (e.g., the E. coli trxB⁻strains) provide cytoplasm conditions that are favorable for disulfidebond formation, thereby permitting proper folding and assembly ofexpressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

The present invention provides an expression system in which thequantitative ratio of expressed polypeptide components can be modulatedin order to maximize the yield of secreted and properly assembledantibodies of the invention. Such modulation is accomplished at least inpart by simultaneously modulating translational strengths for thepolypeptide components.

One technique for modulating translational strength is disclosed inSimmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of thetranslational initiation region (TIR) within a cistron. For a given TIR,a series of amino acid or nucleic acid sequence variants can be createdwith a range of translational strengths, thereby providing a convenientmeans by which to adjust this factor for the desired expression level ofthe specific chain. TIR variants can be generated by conventionalmutagenesis techniques that result in codon changes which can alter theamino acid sequence, although silent changes in the nucleotide sequenceare preferred. Alterations in the TIR can include, for example,alterations in the number or spacing of Shine-Dalgarno sequences, alongwith alterations in the signal sequence. One method for generatingmutant signal sequences is the generation of a “codon bank” at thebeginning of a coding sequence that does not change the amino acidsequence of the signal sequence (i.e., the changes are silent). This canbe accomplished by changing the third nucleotide position of each codon;additionally, some amino acids, such as leucine, serine, and arginine,have multiple first and second positions that can add complexity inmaking the bank. This method of mutagenesis is described in detail inYansura et al. (1992) METHODS: A Companion to Methods in Enzymol.4:151-158.

Preferably, a set of vectors is generated with a range of TIR strengthsfor each cistron therein. This limited set provides a comparison ofexpression levels of each chain as well as the yield of the desiredantibody products under various TIR strength combinations. TIR strengthscan be determined by quantifying the expression level of a reporter geneas described in detail in Simmons et al. U.S. Pat. No. 5,840,523. Basedon the translational strength comparison, the desired individual TIRsare selected to be combined in the expression vector constructs of theinvention.

Prokaryotic host cells suitable for expressing antibodies of theinvention include Archaebacteria and Eubacteria, such as Gram-negativeor Gram-positive organisms. Examples of useful bacteria includeEscherichia (e.g., E. coli), Bacilli (e.g., B. subtilis),Enterobacteria, Pseudomonas species (e.g., P. aeruginosa), Salmonellatyphimurium, Serratia marcescans, Klebsiella, Proteus, Shigella,Rhizobia, Vitreoscilla, or Paracoccus. In one embodiment, gram-negativecells are used. In one embodiment, E. coli cells are used as hosts forthe invention. Examples of E. coli strains include strain W3110(Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.:American Society for Microbiology, 1987), pp. 1190-1219; ATCC DepositNo. 27,325) and derivatives thereof, including strain 33D3 havinggenotype W3110 ΔfhuA (ΔtonA) ptr3 lac Iq lacL8 ΔompTΔ(nmpc-fepE) degP41kan^(R) (U.S. Pat. No. 5,639,635). Other strains and derivativesthereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli _(λ) 1776(ATCC 31,537) and E. coli RV308(ATCC 31,608) are also suitable. Theseexamples are illustrative rather than limiting. Methods for constructingderivatives of any of the above-mentioned bacteria having definedgenotypes are known in the art and described in, for example, Bass etal., Proteins, 8:309-314 (1990). It is generally necessary to select theappropriate bacteria taking into consideration replicability of thereplicon in the cells of a bacterium. For example, E. coli, Serratia, orSalmonella species can be suitably used as the host when well knownplasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supplythe replicon. Typically the host cell should secrete minimal amounts ofproteolytic enzymes, and additional protease inhibitors may desirably beincorporated in the cell culture.

Antibody Production

Host cells are transformed with the above-described expression vectorsand cultured in conventional nutrient media modified as appropriate forinducing promoters, selecting transformants, or amplifying the genesencoding the desired sequences.

Transformation means introducing DNA into the prokaryotic host so thatthe DNA is replicable, either as an extrachromosomal element or bychromosomal integrant. Depending on the host cell used, transformationis done using standard techniques appropriate to such cells. The calciumtreatment employing calcium chloride is generally used for bacterialcells that contain substantial cell-wall barriers. Another method fortransformation employs polyethylene glycol/DMSO. Yet another techniqueused is electroporation.

Prokaryotic cells used to produce the polypeptides of the invention aregrown in media known in the art and suitable for culture of the selectedhost cells. Examples of suitable media include luria broth (LB) plusnecessary nutrient supplements. In some embodiments, the media alsocontains a selection agent, chosen based on the construction of theexpression vector, to selectively permit growth of prokaryotic cellscontaining the expression vector. For example, ampicillin is added tomedia for growth of cells expressing ampicillin resistant gene.

Any necessary supplements besides carbon, nitrogen, and inorganicphosphate sources may also be included at appropriate concentrationsintroduced alone or as a mixture with another supplement or medium suchas a complex nitrogen source. Optionally the culture medium may containone or more reducing agents selected from the group consisting ofglutathione, cysteine, cystamine, thioglycollate, dithioerythritol anddithiothreitol.

The prokaryotic host cells are cultured at suitable temperatures. For E.coli growth, for example, the preferred temperature ranges from about20° C. to about 39° C., more preferably from about 25° C. to about 37°C., even more preferably at about 30° C. The pH of the medium may be anypH ranging from about 5 to about 9, depending mainly on the hostorganism. For E. coli, the pH is preferably from about 6.8 to about 7.4,and more preferably about 7.0.

If an inducible promoter is used in the expression vector of theinvention, protein expression is induced under conditions suitable forthe activation of the promoter. In one aspect of the invention, PhoApromoters are used for controlling transcription of the polypeptides.Accordingly, the transformed host cells are cultured in aphosphate-limiting medium for induction. Preferably, thephosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons etal., J. Immunol. Methods (2002), 263:133-147). A variety of otherinducers may be used, according to the vector construct employed, as isknown in the art.

In one embodiment, the expressed polypeptides of the present inventionare secreted into and recovered from the periplasm of the host cells.Protein recovery typically involves disrupting the microorganism,generally by such means as osmotic shock, sonication or lysis. Oncecells are disrupted, cell debris or whole cells may be removed bycentrifugation or filtration. The proteins may be further purified, forexample, by affinity resin chromatography. Alternatively, proteins canbe transported into the culture media and isolated therein. Cells may beremoved from the culture and the culture supernatant being filtered andconcentrated for further purification of the proteins produced. Theexpressed polypeptides can be further isolated and identified usingcommonly known methods such as polyacrylamide gel electrophoresis (PAGE)and Western blot assay.

In one aspect of the invention, antibody production is conducted inlarge quantity by a fermentation process. Various large-scale fed-batchfermentation procedures are available for production of recombinantproteins. Large-scale fermentations have at least 1000 liters ofcapacity, preferably about 1,000 to 100,000 liters of capacity. Thesefermentors use agitator impellers to distribute oxygen and nutrients,especially glucose (the preferred carbon/energy source). Small scalefermentation refers generally to fermentation in a fermentor that is nomore than approximately 100 liters in volumetric capacity, and can rangefrom about 1 liter to about 100 liters.

In a fermentation process, induction of protein expression is typicallyinitiated after the cells have been grown under suitable conditions to adesired density, e.g., an OD₅₅₀ of about 180-220, at which stage thecells are in the early stationary phase. A variety of inducers may beused, according to the vector construct employed, as is known in the artand described above. Cells may be grown for shorter periods prior toinduction. Cells are usually induced for about 12-50 hours, althoughlonger or shorter induction time may be used.

To improve the production yield and quality of the polypeptides of theinvention, various fermentation conditions can be modified. For example,to improve the proper assembly and folding of the secreted antibodypolypeptides, additional vectors overexpressing chaperone proteins, suchas Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (apeptidylprolyl cis,trans-isomerase with chaperone activity) can be usedto co-transform the host prokaryotic cells. The chaperone proteins havebeen demonstrated to facilitate the proper folding and solubility ofheterologous proteins produced in bacterial host cells. Chen et al.(1999) J Bio Chem 274:19601-19605; Georgiou et al., U.S. Pat. No.6,083,715; Georgiou et al., U.S. Pat. No. 6,027,888; Bothmann andPluckthun (2000) J. Biol. Chem. 275:17100-17105; Ramm and Pluckthun(2000) J. Biol. Chem. 275:17106-17113; Arie et al. (2001) Mol.Microbiol. 39:199-210.

To minimize proteolysis of expressed heterologous proteins (especiallythose that are proteolytically sensitive), certain host strainsdeficient for proteolytic enzymes can be used for the present invention.For example, host cell strains may be modified to effect geneticmutation(s) in the genes encoding known bacterial proteases such asProtease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V,Protease VI and combinations thereof. Some E. coli protease-deficientstrains are available and described in, for example, Joly et al. (1998),supra; Georgiou et al., U.S. Pat. No. 5,264,365; Georgiou et al., U.S.Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2:63-72(1996).

In one embodiment, E. coli strains deficient for proteolytic enzymes andtransformed with plasmids overexpressing one or more chaperone proteinsare used as host cells in the expression system of the invention.

Antibody Purification

In one embodiment, the antibody protein produced herein is furtherpurified to obtain preparations that are substantially homogeneous forfurther assays and uses. Standard protein purification methods known inthe art can be employed. The following procedures are exemplary ofsuitable purification procedures: fractionation on immunoaffinity orion-exchange columns, ethanol precipitation, reverse phase HPLC,chromatography on silica or on a cation-exchange resin such as DEAE,chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gelfiltration using, for example, Sephadex G-75.

In one aspect, Protein A immobilized on a solid phase is used forimmunoaffinity purification of the full length antibody products of theinvention. Protein A is a 41kD cell wall protein from Staphylococcusaureas which binds with a high affinity to the Fc region of antibodies.Lindmark et al (1983) J. Immunol. Meth. 62:1-13. The solid phase towhich Protein A is immobilized is preferably a column comprising a glassor silica surface, more preferably a controlled pore glass column or asilicic acid column. In some applications, the column has been coatedwith a reagent, such as glycerol, in an attempt to prevent nonspecificadherence of contaminants.

As the first step of purification, the preparation derived from the cellculture as described above is applied onto the Protein A immobilizedsolid phase to allow specific binding of the antibody of interest toProtein A. The solid phase is then washed to remove contaminantsnon-specifically bound to the solid phase. Finally the antibody ofinterest is recovered from the solid phase by elution.

Generating Antibodies Using Eukaryotic Host Cells:

The vector components generally include, but are not limited to, one ormore of the following: a signal sequence, an origin of replication, oneor more marker genes, an enhancer element, a promoter, and atranscription termination sequence.

(i) Signal Sequence Component

A vector for use in a eukaryotic host cell may also contain a signalsequence or other polypeptide having a specific cleavage site at theN-terminus of the mature protein or polypeptide of interest. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) by thehost cell. In mammalian cell expression, mammalian signal sequences aswell as viral secretory leaders, for example, the herpes simplex gDsignal, are available.

The DNA for such precursor region is ligated in reading frame to DNAencoding the antibody.

(ii) Origin of Replication

Generally, an origin of replication component is not needed formammalian expression vectors. For example, the SV40 origin may typicallybe used only because it contains the early promoter.

(iii) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, where relevant, or (c) supply critical nutrients notavailable from complex media.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up theantibody nucleic acid, such as DHFR, thymidine kinase, metallothionein-Iand -II, preferably primate metallothionein genes, adenosine deaminase,ornithine decarboxylase, etc.

For example, cells transformed with the DHFR selection gene are firstidentified by culturing all of the transformants in a culture mediumthat contains methotrexate (Mtx), a competitive antagonist of DHFR. Anappropriate host cell when wild-type DHFR is employed is the Chinesehamster ovary (CHO) cell line deficient in DHFR activity (e.g., ATCCCRL-9096).

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody, wild-type DHFR protein, and another selectablemarker such as aminoglycoside 3′-phosphotransferase (APH) can beselected by cell growth in medium containing a selection agent for theselectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

(iv) Promoter Component

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the antibodypolypeptide nucleic acid. Promoter sequences are known for eukaryotes.Virtually alleukaryotic genes have an AT-rich region locatedapproximately 25 to 30 bases upstream from the site where transcriptionis initiated. Another sequence found 70 to 80 bases upstream from thestart of transcription of many genes is a CNCAAT region where N may beany nucleotide. At the 3′ end of most eukaryotic genes is an AATAAAsequence that may be the signal for addition of the poly A tail to the3′ end of the coding sequence. All of these sequences are suitablyinserted into eukaryotic expression vectors.

Antibody polypeptide transcription from vectors in mammalian host cellsis controlled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40(SV40), from heterologous mammalian promoters, e.g., the actin promoteror an immunoglobulin promoter, from heat-shock promoters, provided suchpromoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(v) Enhancer Element Component

Transcription of DNA encoding the antibody polypeptide of this inventionby higher eukaryotes is often increased by inserting an enhancersequence into the vector. Many enhancer sequences are now known frommammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin).Typically, however, one will use an enhancer from a eukaryotic cellvirus. Examples include the SV40 enhancer on the late side of thereplication origin (bp 100-270), the cytomegalovirus early promoterenhancer, the polyoma enhancer on the late side of the replicationorigin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18(1982) on enhancing elements for activation of eukaryotic promoters. Theenhancer may be spliced into the vector at a position 5′ or 3′ to theantibody polypeptide-encoding sequence, but is preferably located at asite 5′ from the promoter.

(vi) Transcription Termination Component

Expression vectors used in eukaryotic host cells will typically alsocontain sequences necessary for the termination of transcription and forstabilizing the mRNA. Such sequences are commonly available from the 5′and, occasionally 3′, untranslated regions of eukaryotic or viral DNAsor cDNAs. These regions contain nucleotide segments transcribed aspolyadenylated fragments in the untranslated portion of the mRNAencoding an antibody. One useful transcription termination component isthe bovine growth hormone polyadenylation region. See WO94/11026 and theexpression vector disclosed therein.

(vii) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein include higher eukaryote cells described herein, includingvertebrate host cells. Propagation of vertebrate cells in culture(tissue culture) has become a routine procedure. Examples of usefulmammalian host cell lines are monkey kidney CV1 line transformed by SV40(COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cellssubcloned for growth in suspension culture, Graham et al., J. Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinesehamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod.23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African greenmonkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinomacells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138,ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor(MMT 060562, ATCC CCL51); TR1 cells (Mather et al., Annals N.Y. Acad.Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatomaline (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(viii) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. No. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(ix) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, or directly secreted into the medium. If the antibodyis produced intracellularly, as a first step, the particulate debris,either host cells or lysed fragments, are removed, for example, bycentrifugation or ultrafiltration. Where the antibody is secreted intothe medium, supernatants from such expression systems are generallyfirst concentrated using a commercially available protein concentrationfilter, for example, an Amicon or Millipore Pellicon ultrafiltrationunit. A protease inhibitor such as PMSF may be included in any of theforegoing steps to inhibit proteolysis and antibiotics may be includedto prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, gel electrophoresis,dialysis, and affinity chromatography, with affinity chromatographybeing the preferred purification technique. The suitability of protein Aas an affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABXTMresin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Following any preliminary purification step(s), the mixture comprisingthe antibody of interest and contaminants may be subjected to low pHhydrophobic interaction chromatography using an elution buffer at a pHbetween about 2.5-4.5, preferably performed at low salt concentrations(e.g., from about 0-0.25M salt).

Activity Assays

The antibodies of the present invention can be characterized for theirphysical/chemical properties and biological functions by various assaysknown in the art.

The purified immunoglobulins can be further characterized by a series ofassays including, but not limited to, N-terminal sequencing, amino acidanalysis, non-denaturing size exclusion high pressure liquidchromatography (HPLC), mass spectrometry, ion exchange chromatographyand papain digestion.

In certain embodiments of the invention, the immunoglobulins producedherein are analyzed for their biological activity. In some embodiments,the immunoglobulins of the present invention are tested for theirantigen binding activity. The antigen binding assays that are known inthe art and can be used herein include without limitation any direct orcompetitive binding assays using techniques such as western blots,radioimmunoassays, ELISA (enzyme linked immunosorbent assay), “sandwich”immunoassays, immunoprecipitation assays, fluorescent immunoassays, andprotein A immunoassays. An illustrative antigen binding assay isprovided below in the Examples section.

In one embodiment, the present invention contemplates an alteredantibody that possesses some but not all effector functions, which makeit a desired candidate for many applications in which the half life ofthe antibody in vivo is important yet certain effector functions (suchas complement and ADCC) are unnecessary or deleterious. In certainembodiments, the Fc activities of the produced immunoglobulin aremeasured to ensure that only the desired properties are maintained. Invitro and/or in vivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). Anexample of an in vitro assay to assess ADCC activity of a molecule ofinterest is described in U.S. Pat. No. 5,500,362 or 5,821,337. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in a animal model such as that disclosed in Clynes et al.PNAS (USA) 95:652-656 (1998). C1q binding assays may also be carried outto confirm that the antibody is unable to bind C1q and hence lacks CDCactivity. To assess complement activation, a CDC assay, e.g. asdescribed in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996),may be performed. FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art,e.g. those described in the Examples section.

Humanized Antibodies

The present invention encompasses humanized antibodies. Various methodsfor humanizing non-human antibodies are known in the art. For example, ahumanized antibody can have one or more amino acid residues introducedinto it from a source which is non-human. These non-human amino acidresidues are often referred to as “import” residues, which are typicallytaken from an “import” variable domain. Humanization can be essentiallyperformed following the method of Winter and co-workers (Jones et al.(1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327;Verhoeyen et al. (1988) Science 239:1534-1536), by substitutingnon-human hypervariable region sequences for the corresponding sequencesof a human antibody. Accordingly, such “humanized” antibodies arechimeric antibodies (U.S. Pat. No. 4,816,567) wherein substantially lessthan an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some hypervariableregion residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies.

The choice of human variable domains, both light and heavy, to be usedin making the humanized antibodies is very important to reduceantigenicity. According to the so-called “best-fit” method, the sequenceof the variable domain of a rodent antibody is screened against theentire library of known human variable-domain sequences. The humansequence which is closest to that of the rodent is then accepted as thehuman framework for the humanized antibody (Sims et al. (1993) J.Immunol. 151:2296; Chothia et al. (1987) J. Mol. Biol. 196:901. Anothermethod uses a particular framework derived from the consensus sequenceof all human antibodies of a particular subgroup of light or heavychains. The same framework may be used for several different humanizedantibodies (Carter et al. (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al. (1993) J. Immunol., 151:2623.

It is further important that antibodies be humanized with retention ofhigh affinity for the antigen and other favorable biological properties.To achieve this goal, according to one method, humanized antibodies areprepared by a process of analysis of the parental sequences and variousconceptual humanized products using three-dimensional models of theparental and humanized sequences. Three-dimensional immunoglobulinmodels are commonly available and are familiar to those skilled in theart. Computer programs are available which illustrate and displayprobable three-dimensional conformational structures of selectedcandidate immunoglobulin sequences. Inspection of these displays permitsanalysis of the likely role of the residues in the functioning of thecandidate immunoglobulin sequence, i.e., the analysis of residues thatinfluence the ability of the candidate immunoglobulin to bind itsantigen. In this way, FR residues can be selected and combined from therecipient and import sequences so that the desired antibodycharacteristic, such as increased affinity for the target antigen(s), isachieved. In general, the hypervariable region residues are directly andmost substantially involved in influencing antigen binding.

Antibody Variants

In one aspect, the invention provides antibody fragment comprisingmodifications in the interface of Fc polypeptides comprising the Fcregion, wherein the modifications facilitate and/or promoteheterodimerization. These modifications comprise introduction of aprotuberance into a first Fc polypeptide and a cavity into a second Fcpolypeptide, wherein the protuberance is positionable in the cavity soas to promote complexing of the first and second Fc polypeptides.Methods of generating antibodies with these modifications are known inthe art, e.g., as described in U.S. Pat. No. 5,731,168.

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodyare prepared by introducing appropriate nucleotide changes into theantibody nucleic acid, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of, residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution ismade to arrive at the final construct, provided that the final constructpossesses the desired characteristics. The amino acid alterations may beintroduced in the subject antibody amino acid sequence at the time thatsequence is made.

A useful method for identification of certain residues or regions of theantibody that are preferred locations for mutagenesis is called “alaninescanning mutagenesis” as described by Cunningham and Wells (1989)Science, 244:1081-1085. Here, a residue or group of target residues areidentified (e.g., charged residues such as arg, asp, his, lys, and glu)and replaced by a neutral or negatively charged amino acid (mostpreferably alanine or polyalanine) to affect the interaction of theamino acids with antigen. Those amino acid locations demonstratingfunctional sensitivity to the substitutions then are refined byintroducing further or other variants at, or for, the sites ofsubstitution. Thus, while the site for introducing an amino acidsequence variation is predetermined, the nature of the mutation per seneed not be predetermined. For example, to analyze the performance of amutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressedimmunoglobulins are screened for the desired activity.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue or the antibody fusedto a cytotoxic polypeptide. Other insertional variants of the antibodymolecule include the fusion to the N- or C-terminus of the antibody toan enzyme (e.g. for ADEPT) or a polypeptide which increases the serumhalf-life of the antibody.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculereplaced by a different residue. The sites of greatest interest forsubstitutional mutagenesis include the hypervariable regions, but FRalterations are also contemplated. Conservative substitutions are shownin Table 2 under the heading of “preferred substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 2,or as further described below in reference to amino acid classes, may beintroduced and the products screened.

TABLE 2 Original Exemplary Preferred Residue Substitutions SubstitutionsAla (A) Val; Leu; Ile Val Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His;Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn;Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; ArgArg Ile (I) Leu; Val; Met; Ala; Leu Phe; Norleucine Leu (L) Norleucine;Ile; Val; Ile Met; Ala; Phe Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe;Ile Leu Phe (F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S)Thr Thr Thr (T) Val; Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr;Ser Phe Val (V) Ile; Leu; Met; Phe; Leu Ala; Norleucine

Substantial modifications in the biological properties of the antibodyare accomplished by selecting substitutions that differ significantly intheir effect on maintaining (a) the structure of the polypeptidebackbone in the area of the substitution, for example, as a sheet orhelical conformation, (b) the charge or hydrophobicity of the moleculeat the target site, or (c) the bulk of the side chain. Amino acids maybe grouped according to similarities in the properties of their sidechains (in A. L. Lehninger, in Biochemistry, second ed., pp. 73-75,Worth Publishers, New York (1975)):

(1) non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp(W), Met (M)(2) uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn(N), Gln (Q)(3) acidic: Asp (D), Glu (E)(4) basic: Lys (K), Arg (R), H is(H)

Alternatively, naturally occurring residues may be divided into groupsbased on common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

(3) acidic: Asp, Glu;

(4) basic: H is, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class. Such substituted residues also may beintroduced into the conservative substitution sites or, more preferably,into the remaining (non-conserved) sites.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther development will have improved biological properties relative tothe parent antibody from which they are generated. A convenient way forgenerating such substitutional variants involves affinity maturationusing phage display. Briefly, several hypervariable region sites (e.g.6-7 sites) are mutated to generate all possible amino acid substitutionsat each site. The antibodies thus generated are displayed fromfilamentous phage particles as fusions to the gene III product of M13packaged within each particle. The phage-displayed variants are thenscreened for their biological activity (e.g. binding affinity) as hereindisclosed. In order to identify candidate hypervariable region sites formodification, alanine scanning mutagenesis can be performed to identifyhypervariable region residues contributing significantly to antigenbinding. Alternatively, or additionally, it may be beneficial to analyzea crystal structure of the antigen-antibody complex to identify contactpoints between the antibody and antigen. Such contact residues andneighboring residues are candidates for substitution according to thetechniques elaborated herein. Once such variants are generated, thepanel of variants is subjected to screening as described herein andantibodies with superior properties in one or more relevant assays maybe selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of theantibody are prepared by a variety of methods known in the art. Thesemethods include, but are not limited to, isolation from a natural source(in the case of naturally occurring amino acid sequence variants) orpreparation by oligonucleotide-mediated (or site-directed) mutagenesis,PCR mutagenesis, and cassette mutagenesis of an earlier prepared variantor a non-variant version of the antibody.

It may be desirable to introduce one or more amino acid modifications inan Fc region of the immunoglobulin polypeptides of the invention,thereby generating a Fc region variant. The Fc region variant maycomprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 orIgG4 Fc region) comprising an amino acid modification (e.g. asubstitution) at one or more amino acid positions including that of ahinge cysteine.

In accordance with this description and the teachings of the art, it iscontemplated that in some embodiments, an antibody used in methods ofthe invention may comprise one or more alterations as compared to thewild type counterpart antibody, e.g. in the Fc region. These antibodieswould nonetheless retain substantially the same characteristics requiredfor therapeutic utility as compared to their wild type counterpart. Forexample, it is thought that certain alterations can be made in the Fcregion that would result in altered (i.e., either improved ordiminished) C1q binding and/or Complement Dependent Cytotoxicity (CDC),e.g., as described in WO99/51642. See also Duncan & Winter Nature322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; andWO94/29351 concerning other examples of Fc region variants.

Immunoconjugates

The invention also pertains to immunoconjugates, or antibody-drugconjugates (ADC), comprising an antibody conjugated to a cytotoxic agentsuch as a chemotherapeutic agent, a drug, a growth inhibitory agent, atoxin (e.g., an enzymatically active toxin of bacterial, fungal, plant,or animal origin, or fragments thereof), or a radioactive isotope (i.e.,a radioconjugate).

The use of antibody-drug conjugates for the local delivery of cytotoxicor cytostatic agents, i.e. drugs to kill or inhibit tumor cells in thetreatment of cancer (Syrigos and Epenetos (1999) Anticancer Research19:605-614; Niculescu-Duvaz and Springer (1997)

Adv. Drg Del. Rev. 26:151-172; U.S. Pat. No. 4,975,278) theoreticallyallows targeted delivery of the drug moiety to tumors, and intracellularaccumulation therein, where systemic administration of theseunconjugated drug agents may result in unacceptable levels of toxicityto normal cells as well as the tumor cells sought to be eliminated(Baldwin et al., (1986) Lancet pp. (Mar. 15, 1986):603-05; Thorpe,(1985) “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: AReview,” in Monoclonal Antibodies '84: Biological And ClinicalApplications, A. Pinchera et al. (ed.$), pp. 475-506). Maximal efficacywith minimal toxicity is sought thereby. Both polyclonal antibodies andmonoclonal antibodies have been reported as useful in these strategies(Rowland et al., (1986) Cancer Immunol. Immunother., 21:183-87). Drugsused in these methods include daunomycin, doxorubicin, methotrexate, andvindesine (Rowland et al., (1986) supra). Toxins used in antibody-toxinconjugates include bacterial toxins such as diphtheria toxin, planttoxins such as ricin, small molecule toxins such as geldanamycin(Mandler et al (2000) Jour. of the Nat. Cancer Inst. 92(19):1573-1581;Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028;Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP1391213; Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623),and calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al(1993) Cancer Res. 53:3336-3342). The toxins may effect their cytotoxicand cytostatic effects by mechanisms including tubulin binding, DNAbinding, or topoisomerase inhibition. Some cytotoxic drugs tend to beinactive or less active when conjugated to large antibodies or proteinreceptor ligands.

ZEVALIN® (ibritumomab tiuxetan, Biogen/Idec) is an antibody-radioisotopeconjugate composed of a murine IgG1 kappa monoclonal antibody directedagainst the CD20 antigen found on the surface of normal and malignant Blymphocytes and ¹¹¹In or ⁹⁰Y radioisotope bound by a thiourealinker-chelator (Wiseman et al (2000) Eur. Jour. Nucl. Med.27(7):766-77; Wiseman et al (2002) Blood 99(12):4336-42; Witzig et al(2002) J. Clin. Oncol. 20(10):2453-63; Witzig et al (2002) J. Clin.Oncol. 20(15):3262-69). Although ZEVALIN has activity against B-cellnon-Hodgkin's Lymphoma (NHL), administration results in severe andprolonged cytopenias in most patients. MYLOTARG™ (gemtuzumab ozogamicin,Wyeth Pharmaceuticals), an antibody drug conjugate composed of a hu CD33antibody linked to calicheamicin, was approved in 2000 for the treatmentof acute myeloid leukemia by injection (Drugs of the Future (2000)25(7):686; U.S. Pat. Nos. 4,970,198; 5,079,233; 5,585,089; 5,606,040;5,693,762; 5,739,116; 5,767,285; 5,773,001). Cantuzumab mertansine(Immunogen, Inc.), an antibody drug conjugate composed of the huC242antibody linked via the disulfide linker SPP to the maytansinoid drugmoiety, DM1, is advancing into Phase II trials for the treatment ofcancers that express CanAg, such as colon, pancreatic, gastric, andothers. MLN-2704 (Millennium Pharm., BZL Biologics, Immunogen Inc.), anantibody drug conjugate composed of the anti-prostate specific membraneantigen (PSMA) monoclonal antibody linked to the maytansinoid drugmoiety, DM1, is under development for the potential treatment ofprostate tumors. The auristatin peptides, auristatin E (AE) andmonomethylauristatin (MMAE), synthetic analogs of dolastatin, wereconjugated to chimeric monoclonal antibodies cBR96 (specific to Lewis Yon carcinomas) and cAC10 (specific to CD30 on hematologicalmalignancies) (Doronina et al (2003) Nature Biotechnology 21(7):778-784)and are under therapeutic development.

Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate),aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026.

Conjugates of an antibody and one or more small molecule toxins, such asa calicheamicin, maytansinoids, a trichothecene, and CC 1065, and thederivatives of these toxins that have toxin activity, are alsocontemplated herein.

Maytansine and Maytansinoids

In one embodiment, an antibody (full length or fragments) of theinvention is conjugated to one or more maytansinoid molecules.

Maytansinoids are mitototic inhibitors which act by inhibiting tubulinpolymerization. Maytansine was first isolated from the east Africanshrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it wasdiscovered that certain microbes also produce maytansinoids, such asmaytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042).Synthetic maytansinol and derivatives and analogues thereof aredisclosed, for example, in U.S. Pat. Nos. 4,137,230; 4,248,870;4,256,746; 4,260,608; 4,265,814; 4,294,757; 4,307,016; 4,308,268;4,308,269; 4,309,428; 4,313,946; 4,315,929; 4,317,821; 4,322,348;4,331,598; 4,361,650; 4,364,866; 4,424,219; 4,450,254; 4,362,663; and4,371,533, the disclosures of which are hereby expressly incorporated byreference.

Maytansinoid-Antibody Conjugates

In an attempt to improve their therapeutic index, maytansine andmaytansinoids have been conjugated to antibodies specifically binding totumor cell antigens. Immunoconjugates containing maytansinoids and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020,5,416,064 and European Patent EP 0 425 235 B1, the disclosures of whichare hereby expressly incorporated by reference. Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-8623 (1996) described immunoconjugates comprisinga maytansinoid designated DM1 linked to the monoclonal antibody C242directed against human colorectal cancer. The conjugate was found to behighly cytotoxic towards cultured colon cancer cells, and showedantitumor activity in an in vivo tumor growth assay. Chari et al.,Cancer Research 52:127-131 (1992) describe immunoconjugates in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×10⁵ HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Antibody-Maytansinoid Conjugates (Immunoconjugates)

Antibody-maytansinoid conjugates are prepared by chemically linking anantibody to a maytansinoid molecule without significantly diminishingthe biological activity of either the antibody or the maytansinoidmolecule. An average of 3-4 maytansinoid molecules conjugated perantibody molecule has shown efficacy in enhancing cytotoxicity of targetcells without negatively affecting the function or solubility of theantibody, although even one molecule of toxin/antibody would be expectedto enhance cytotoxicity over the use of naked antibody. Maytansinoidsare well known in the art and can be synthesized by known techniques orisolated from natural sources. Suitable maytansinoids are disclosed, forexample, in U.S. Pat. No. 5,208,020 and in the other patents andnonpatent publications referred to hereinabove. Preferred maytansinoidsare maytansinol and maytansinol analogues modified in the aromatic ringor at other positions of the maytansinol molecule, such as variousmaytansinol esters.

There are many linking groups known in the art for makingantibody-maytansinoid conjugates, including, for example, thosedisclosed in U.S. Pat. No. 5,208,020 or EP Patent 0 425 235 B1, andChari et al., Cancer Research 52:127-131 (1992). The linking groupsinclude disulfide groups, thioether groups, acid labile groups,photolabile groups, peptidase labile groups, or esterase labile groups,as disclosed in the above-identified patents, disulfide and thioethergroups being preferred.

Conjugates of the antibody and maytansinoid may be made using a varietyof bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). Particularly preferred coupling agentsinclude N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP) (Carlssonet al., Biochem. J. 173:723-737 [1978]) andN-succinimidyl-4-(2-pyridylthio)pentanoate (SPP) to provide for adisulfide linkage.

The linker may be attached to the maytansinoid molecule at variouspositions, depending on the type of the link. For example, an esterlinkage may be formed by reaction with a hydroxyl group usingconventional coupling techniques. The reaction may occur at the C-3position having a hydroxyl group, the C-14 position modified withhydroxymethyl, the C-15 position modified with a hydroxyl group, and theC-20 position having a hydroxyl group. In a preferred embodiment, thelinkage is formed at the C-3 position of maytansinol or a maytansinolanalogue.

Calicheamicin

Another immunoconjugate of interest comprises an antibody conjugated toone or more calicheamicin molecules. The calicheamicin family ofantibiotics are capable of producing double-stranded DNA breaks atsub-picomolar concentrations. For the preparation of conjugates of thecalicheamicin family, see U.S. Pat. Nos. 5,712,374, 5,714,586,5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, 5,877,296 (all toAmerican Cyanamid Company). Structural analogues of calicheamicin whichmay be used include, but are not limited to, γ₁ ^(I), α₂ ^(I), α₃ ^(I),N-acetyl-γ₁ ^(I), PSAG and θ^(I) ₁ (Hinman et al., Cancer Research53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998)and the aforementioned U.S. patents to American Cyanamid). Anotheranti-tumor drug that the antibody can be conjugated is QFA which is anantifolate. Both calicheamicin and QFA have intracellular sites ofaction and do not readily cross the plasma membrane. Therefore, cellularuptake of these agents through antibody mediated internalization greatlyenhances their cytotoxic effects.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an immunoconjugate formedbetween an antibody and a compound with nucleolytic activity (e.g., aribonuclease or a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt²¹¹, I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² andradioactive isotopes of Lu. When the conjugate is used for detection, itmay comprise a radioactive atom for scintigraphic studies, for exampletc^(99m) or I¹²³, or a spin label for nuclear magnetic resonance (NMR)imaging (also known as magnetic resonance imaging, mri), such asiodine-123 again, iodine-131, indium-111, fluorine-19, carbon-13,nitrogen-15, oxygen-17, gadolinium, manganese or iron.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as tc^(99m) or I¹²³, Re¹⁸⁶, Re¹⁸⁸ and In¹¹¹ can be attachedvia a cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun. 80: 49-57 can be used to incorporate iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

Conjugates of the antibody and cytotoxic agent may be made using avariety of bifunctional protein coupling agents such asN-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate,iminothiolane (IT), bifunctional derivatives of imidoesters (such asdimethyl adipimidate HCl), active esters (such as disuccinimidylsuberate), aldehydes (such as glutaraldehyde), bis-azido compounds (suchas bis (p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (suchas bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238:1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO94/11026. Thelinker may be a “cleavable linker” facilitating release of the cytotoxicdrug in the cell. For example, an acid-labile linker,peptidase-sensitive linker, photolabile linker, dimethyl linker ordisulfide-containing linker (Chari et al., Cancer Research 52:127-131(1992); U.S. Pat. No. 5,208,020) may be used.

The compounds of the invention expressly contemplate, but are notlimited to, ADC prepared with cross-linker reagents: BMPS, EMCS, GMBS,HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS,sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, andsulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) which arecommercially available (e.g., from Pierce Biotechnology, Inc., Rockford,Ill., U.S.A). See pages 467-498, 2003-2004 Applications Handbook andCatalog.

Preparation of Antibody Drug Conjugates

In the antibody drug conjugates (ADC) of the invention, an antibody (Ab)is conjugated to one or more drug moieties (D), e.g. about 1 to about 20drug moieties per antibody, through a linker (L). The ADC of Formula Imay be prepared by several routes, employing organic chemistryreactions, conditions, and reagents known to those skilled in the art,including: (1) reaction of a nucleophilic group of an antibody with abivalent linker reagent, to form Ab-L, via a covalent bond, followed byreaction with a drug moiety D; and (2) reaction of a nucleophilic groupof a drug moiety with a bivalent linker reagent, to form D-L, via acovalent bond, followed by reaction with the nucleophilic group of anantibody.

Ab-(L-D)_(p)  I

Nucleophilic groups on antibodies include, but are not limited to: (i)N-terminal amine groups, (ii) side chain amine groups, e.g. lysine,(iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl oramino groups where the antibody is glycosylated. Amine, thiol, andhydroxyl groups are nucleophilic and capable of reacting to formcovalent bonds with electrophilic groups on linker moieties and linkerreagents including: (i) active esters such as NHS esters, HOBt esters,haloformates, and acid halides; (ii) alkyl and benzyl halides such ashaloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimidegroups. Certain antibodies have reducible interchain disulfides, i.e.cysteine bridges. Antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(dithiothreitol). Each cysteine bridge will thus form, theoretically,two reactive thiol nucleophiles. Additional nucleophilic groups can beintroduced into antibodies through the reaction of lysines with2-iminothiolane (Traut's reagent) resulting in conversion of an amineinto a thiol.

Antibody drug conjugates of the invention may also be produced bymodification of the antibody to introduce electrophilic moieties, whichcan react with nucleophilic substituents on the linker reagent or drug.The sugars of glycosylated antibodies may be oxidized, e.g. withperiodate oxidizing reagents, to form aldehyde or ketone groups whichmay react with the amine group of linker reagents or drug moieties. Theresulting imine Schiff base groups may form a stable linkage, or may bereduced, e.g. by borohydride reagents to form stable amine linkages. Inone embodiment, reaction of the carbohydrate portion of a glycosylatedantibody with either glactose oxidase or sodium meta-periodate may yieldcarbonyl (aldehyde and ketone) groups in the protein that can react withappropriate groups on the drug (Hermanson, Bioconjugate Techniques). Inanother embodiment, proteins containing N-terminal serine or threonineresidues can react with sodium meta-periodate, resulting in productionof an aldehyde in place of the first amino acid (Geoghegan & Stroh,(1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Suchaldehyde can be reacted with a drug moiety or linker nucleophile.

Likewise, nucleophilic groups on a drug moiety include, but are notlimited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine,thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groupscapable of reacting to form covalent bonds with electrophilic groups onlinker moieties and linker reagents including: (i) active esters such asNHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups.

Alternatively, a fusion protein comprising the antibody and cytotoxicagent may be made, e.g., by recombinant techniques or peptide synthesis.The length of DNA may comprise respective regions encoding the twoportions of the conjugate either adjacent one another or separated by aregion encoding a linker peptide which does not destroy the desiredproperties of the conjugate.

In yet another embodiment, the antibody may be conjugated to a“receptor” (such streptavidin) for utilization in tumor pre-targetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin)which is conjugated to a cytotoxic agent (e.g., a radionucleotide).

Antibody Derivatives

The antibodies of the present invention can be further modified tocontain additional nonproteinaceous moieties that are known in the artand readily available. Preferably, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymers are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

Pharmaceutical Formulations

Therapeutic formulations comprising an antibody of the invention areprepared for storage by mixing the antibody having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of aqueous solutions, lyophilized or otherdried formulations. Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed, andinclude buffers such as phosphate, citrate, histidine and other organicacids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g., Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the immunoglobulin of the invention,which matrices are in the form of shaped articles, e.g., films, ormicrocapsule. Examples of sustained-release matrices include polyesters,hydrogels (for example, poly(2-hydroxyethyl-methacrylate), orpoly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymersof L-glutamic acid and γ ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (injectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods. When encapsulated immunoglobulins remain in the body for a longtime, they may denature or aggregate as a result of exposure to moistureat 37° C., resulting in a loss of biological activity and possiblechanges in immunogenicity. Rational strategies can be devised forstabilization depending on the mechanism involved. For example, if theaggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization may beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

Uses

An antibody of the present invention may be used in, for example, invitro, ex vivo and in vivo therapeutic methods. Antibodies of theinvention can be used as an antagonist to partially or fully block thespecific antigen activity in vitro, ex vivo and/or in vivo. Moreover, atleast some of the antibodies of the invention can neutralize antigenactivity from other species. Accordingly, the antibodies of theinvention can be used to inhibit a specific antigen activity, e.g., in acell culture containing the antigen, in human subjects or in othermammalian subjects having the antigen with which an antibody of theinvention cross-reacts (e.g. chimpanzee, baboon, marmoset, cynomolgusand rhesus, pig or mouse). In one embodiment, the antibody of theinvention can be used for inhibiting antigen activities by contactingthe antibody with the antigen such that antigen activity is inhibited.Preferably, the antigen is a human protein molecule.

In one embodiment, an antibody of the invention can be used in a methodfor inhibiting an antigen in a subject suffering from a disorder inwhich the antigen activity is detrimental, comprising administering tothe subject an antibody of the invention such that the antigen activityin the subject is inhibited. Preferably, the antigen is a human proteinmolecule and the subject is a human subject. Alternatively, the subjectcan be a mammal expressing the antigen with which an antibody of theinvention binds. Still further the subject can be a mammal into whichthe antigen has been introduced (e.g., by administration of the antigenor by expression of an antigen transgene). An antibody of the inventioncan be administered to a human subject for therapeutic purposes.Moreover, an antibody of the invention can be administered to anon-human mammal expressing an antigen with which the immunoglobulincross-reacts (e.g., a primate, pig or mouse) for veterinary purposes oras an animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy ofantibodies of the invention (e.g., testing of dosages and time coursesof administration). Blocking antibodies of the invention that aretherapeutically useful include, for example but are not limited to,anti-HER2, anti-VEGF, anti-IgE, anti-CD11, anti-interferon andanti-tissue factor antibodies. The antibodies of the invention can beused to treat, inhibit, delay progression of, prevent/delay recurrenceof, ameliorate, or prevent diseases, disorders or conditions associatedwith abnormal expression and/or activity of one or more antigenmolecules, including but not limited to malignant and benign tumors;non-leukemias and lymphoid malignancies; neuronal, glial, astrocytal,hypothalamic and other glandular, macrophagal, epithelial, stromal andblastocoelic disorders; and inflammatory, angiogenic and immunologicdisorders.

In one aspect, a blocking antibody of the invention is specific to aligand antigen, and inhibits the antigen activity by blocking orinterfering with the ligand-receptor interaction involving the ligandantigen, thereby inhibiting the corresponding signal pathway and othermolecular or cellular events. The invention also featuresreceptor-specific antibodies which do not necessarily prevent ligandbinding but interfere with receptor activation, thereby inhibiting anyresponses that would normally be initiated by the ligand binding. Theinvention also encompasses antibodies that either preferably orexclusively bind to ligand-receptor complexes. An antibody of theinvention can also act as an agonist of a particular antigen receptor,thereby potentiating, enhancing or activating either all or partialactivities of the ligand-mediated receptor activation.

In certain embodiments, an immunoconjugate comprising an antibodyconjugated with a cytotoxic agent is administered to the patient. Insome embodiments, the immunoconjugate and/or antigen to which it isbound is/are internalized by the cell, resulting in increasedtherapeutic efficacy of the immunoconjugate in killing the target cellto which it binds. In one embodiment, the cytotoxic agent targets orinterferes with nucleic acid in the target cell. Examples of suchcytotoxic agents include any of the chemotherapeutic agents noted herein(such as a maytansinoid or a calicheamicin), a radioactive isotope, or aribonuclease or a DNA endonuclease.

Antibodies of the invention can be used either alone or in combinationwith other compositions in a therapy. For instance, an antibody of theinvention may be co-administered with another antibody, chemotherapeuticagent(s) (including cocktails of chemotherapeutic agents), othercytotoxic agent(s), anti-angiogenic agent(s), cytokines, and/or growthinhibitory agent(s). Where an antibody of the invention inhibits tumorgrowth, it may be particularly desirable to combine it with one or moreother therapeutic agent(s) which also inhibits tumor growth. Forinstance, an antibody of the invention may be combined with an anti-VEGFantibody (e.g., AVASTIN) and/or anti-ErbB antibodies (e.g. HERCEPTIN®anti-HER2 antibody) in a treatment scheme, e.g. in treating any of thediseases described herein, including colorectal cancer, metastaticbreast cancer and kidney cancer. Alternatively, or additionally, thepatient may receive combined radiation therapy (e.g. external beamirradiation or therapy with a radioactive labeled agent, such as anantibody). Such combined therapies noted above include combinedadministration (where the two or more agents are included in the same orseparate formulations), and separate administration, in which case,administration of the antibody of the invention can occur prior to,and/or following, administration of the adjunct therapy or therapies.

The antibody of the invention (and adjunct therapeutic agent) is/areadministered by any suitable means, including parenteral, subcutaneous,intraperitoneal, intrapulmonary, and intranasal, and, if desired forlocal treatment, intralesional administration. Parenteral infusionsinclude intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. In addition, the antibody is suitablyadministered by pulse infusion, particularly with declining doses of theantibody. Dosing can be by any suitable route, e.g. by injections, suchas intravenous or subcutaneous injections, depending in part on whetherthe administration is brief or chronic.

The antibody composition of the invention will be formulated, dosed, andadministered in a fashion consistent with good medical practice. Factorsfor consideration in this context include the particular disorder beingtreated, the particular mammal being treated, the clinical condition ofthe individual patient, the cause of the disorder, the site of deliveryof the agent, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Theantibody need not be, but is optionally formulated with one or moreagents currently used to prevent or treat the disorder in question. Theeffective amount of such other agents depends on the amount ofantibodies of the invention present in the formulation, the type ofdisorder or treatment, and other factors discussed above. These aregenerally used in the same dosages and with administration routes asused hereinbefore or about from 1 to 99% of the heretofore employeddosages.

For the prevention or treatment of disease, the appropriate dosage of anantibody of the invention (when used alone or in combination with otheragents such as chemotherapeutic agents) will depend on the type ofdisease to be treated, the type of antibody, the severity and course ofthe disease, whether the antibody is administered for preventive ortherapeutic purposes, previous therapy, the patient's clinical historyand response to the antibody, and the discretion of the attendingphysician. The antibody is suitably administered to the patient at onetime or over a series of treatments. Depending on the type and severityof the disease, about 1 μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) ofantibody is an initial candidate dosage for administration to thepatient, whether, for example, by one or more separate administrations,or by continuous infusion. One typical daily dosage might range fromabout 1 μg/kg to 100 mg/kg or more, depending on the factors mentionedabove. For repeated administrations over several days or longer,depending on the condition, the treatment is sustained until a desiredsuppression of disease symptoms occurs. One exemplary dosage of theantibody would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, e.g. about six doses of the antibody). An initial higher loadingdose, followed by one or more lower doses may be administered. Anexemplary dosing regimen comprises administering an initial loading doseof about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kgof the antibody. However, other dosage regimens may be useful. Theprogress of this therapy is easily monitored by conventional techniquesand assays.

Articles of Manufacture

In another aspect of the invention, an article of manufacture containingmaterials useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacturecomprises a container and a label or package insert on or associatedwith the container. Suitable containers include, for example, bottles,vials, syringes, etc. The containers may be formed from a variety ofmaterials such as glass or plastic. The container holds a compositionwhich is by itself or when combined with another composition effectivefor treating, preventing and/or diagnosing the condition and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). At least one active agent in the composition is anantibody of the invention. The label or package insert indicates thatthe composition is used for treating the condition of choice, such ascancer. Moreover, the article of manufacture may comprise (a) a firstcontainer with a composition contained therein, wherein the compositioncomprises an antibody of the invention; and (b) a second container witha composition contained therein, wherein the composition comprises afurther cytotoxic agent. The article of manufacture in this embodimentof the invention may further comprise a package insert indicating thatthe first and second antibody compositions can be used to treat aparticular condition, e.g. cancer. Alternatively, or additionally, thearticle of manufacture may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

The following are examples of the methods and compositions of theinvention. It is understood that various other embodiments may bepracticed, given the general description provided above.

EXAMPLES Materials and Methods

Residue numbers are according to Kabat (Kabat et al., Sequences ofproteins of immunological interest, 5th Ed., Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Single letteramino acid abbreviations are used. DNA degeneracies are representedusing the IUB code (N=A/C/G/T, D=A/G/T, V=A/C/G, B=C/G/T, H=A/C/T,K=G/T, M=A/C, R=A/G, S=G/C, W=A/T, Y═C/T).

Direct hypervariable region grafts onto the acceptor human consensusframework—The phagemid used for this work is a monovalent Fab-g3 displayvector (pV0350-2B) having 2 open reading frames under control of thephoA promoter, essentially as described in Lee et al., J. Mol. Biol.(2004), 340(5):1073-93. The first open reading frame consists of thestII signal sequence fused to the VL and CL domains of the acceptorlight chain and the second consists of the stII signal sequence fused tothe VH and CH1 domains of the acceptor heavy chain followed by atruncated minor phage coat protein P3. See Lee et al., supra.

The VL and VH domains from murine 5D5 (see hybridoma 5D5.11.6, ATCCDeposit No. HB-11895, deposit date May 23, 1995) were aligned with thehuman consensus kappa I (huKI) and human subgroup III consensus VH(huIII) domains. To make the HVR graft, the acceptor VH framework, whichdiffers from the human subgroup III consensus VH domain at 3 positions:R71A, N73T, and L78A (Carter et al., Proc. Natl. Acad. Sci. USA 89:4285(1992)) was used. Hypervariable regions from the murine 5D5 (mu5D5)antibody were engineered into the acceptor human consensus framework togenerate a direct HVR-graft of 5D5 (5D5 graft). In the VL domain thefollowing regions were grafted to the human consensus acceptor:positions 24-34 (L1), 50-56 (L2) and 89-97 (L3). In the VH domain,positions 26-35 (H1), 49-65 (H2) and 95-102 (H3) were grafted (FIG. 1).

The direct-graft variants were generated by Kunkel mutagenesis using aseparate oligonucleotide for each hypervariable region. Correct cloneswere assessed by DNA sequencing.

Soft randomization of the hypervariable regions—Sequence diversity wasintroduced into each hypervariable region using a soft randomizationstrategy that maintains a bias towards the murine hypervariable regionsequence. This was accomplished using a poisoned oligonucleotidesynthesis strategy as described by Gallop et al., J. Med. Chem.37:1233-1251 (1994). For a given position within a hypervariable regionto be mutated, the codon encoding the wild-type amino acid is poisonedwith a 70-10-10-10 mixture of nucleotides resulting in an average 50percent mutation rate at each position.

Soft randomized oligonucleotides were patterned after the murinehypervariable region sequences and encompassed the same regions definedby the direct hypervariable region grafts. The amino acid position atthe beginning of H2 (position 49) in the VH domain, was limited insequence diversity to A, G, S or T by using the codon RGC.

Generation of phage libraries—Randomized oligonucleotide pools designedfor each hypervariable region were phoshorylated separately in six 20 μlreactions containing 660 ng of oligonucleotide, 50 mM Tris pH 7.5, 10 mMMgCl₂, 1 mM ATP, 20 mM DTT, and 5 U polynucleotide kinase for 1 h at 37°C. The six phosphorylated oligonucleotide pools were then combined with20 μg of Kunkel template in 50 mM Tris pH 7.5, 10 mM MgCl₂ in a finalvolume of 500 μl resulting in a oligonucleotide to template ratio of 3.The mixture was annealed at 90° C. for 4 min, 50° C. for 5 min and thencooled on ice. Excess, unannealed oligonucleotide was removed with aQIAQUICK PCR purification kit (Qiagen kit 28106) using a modifiedprotocol to prevent excessive denaturation of the annealed DNA. To the500 μl of annealed mixture, 150 μl of PB was added, and the mixture wassplit between 2 silica columns. Following a wash of each column with 750μl of PE and an extra spin to dry the columns, each column was elutedwith 110 μl of 10 mM Tris, 1 mM EDTA, pH 8. The annealed and cleaned-uptemplate (220 μl) was then filled in by adding 1 μl 100 mM ATP, 10 μl 25mM dNTPs (25 mM each of dATP, dCTP, dGTP and dTTP), 15 μl 100 mM DTT, 25μl 10×TM buffer (0.5 M Tris pH 7.5, 0.1 M MgCl₂), 2400 U T4 ligase, and30 U T7 polymerase for 3 h at room temperature.

The filled in product was analyzed on Tris-Acetate-EDTA/agarose gels(Sidhu et al., Methods in Enzymology 328:333-363 (2000)). Three bandswere usually visible: the bottom band is a correctly filled and ligatedproduct, the middle band is a filled but unligated product, and the topband is a strand displaced product. The top band is produced by anintrinsic side activity of T7 polymerase and is difficult to avoid(Lechner et al., J. Biol. Chem. 258:11174-11184 (1983)); however, thisband transforms 30-fold less efficiently than the bottom band andusually contributes little to the library. The middle band is due to theabsence of a 5′ phosphate for the final ligation reaction; this bandtransforms efficiently and gives mainly wild type sequence.

The filled in product was then cleaned-up and electroporated into SS320cells and propagated in the presence of M13/KO7 helper phage asdescribed by Sidhu et al., Methods in Enzymology 328:333-363 (2000).Library sizes ranged from 1-2×10⁹ independent clones. Random clones fromthe initial libraries were sequenced to assess library quality.

Phage Selection—The human HGF receptor was generated and used as an Fcfusion (HGFR-Fc) (Mark et al., J. Biol. Chem. (1992), 267:26166-26171).HGFR-Fc was coated on MaxiSorp microtiter plates (Nunc) at 5 μg/ml inPBS. For the first round of selection 8 wells of target were used; asingle well of target was used for successive rounds of selection. Wellswere blocked for 1 h using Casein Blocker (Pierce). Phage were harvestedfrom the culture supernatant and suspended in PBS containing 1% BSA and0.05% TWEEN 20 (PBSBT). After binding to the wells for 2 h, unboundphage were removed by extensive washing with PBS containing 0.05% TWEEN20 (PBST). Bound phage were eluted by incubating the wells with 50 mMHCl, 0.5 M KCl for 30 min. Phage were amplified using Top10 cells andM13/KO7 helper phage and grown overnight at 37° C. in 2YT, 50 μg/mlcarbenecillin. The titers of phage eluted from a target coated well werecompared to titers of phage recovered from a non-target coated well toassess enrichment.

For affinity maturation, phage libraries were sorted using a solutionsorting method. HFGR-Fc was biotinylated by mixing 500 μl of 3.6 mg/mlHGFR-Fc in PBS, and 10 μl of 1 M Potassium phosphate, pH 8 with 20 μl 4mM Sulfo-NHS-LC-biotin (Pierce). Biotinylated HGFR-Fc (b-HGFR-Fc) waspurified using a NAPS column (Amersham Biosciences) in PBS. Microtiterwells were coated with 10 μg/ml neutravidin in PBS overnight at 4° C.and then blocked for 1 h using Casein Blocker (Pierce). In the firstround of panning, 200 μl phage suspended in PBS containing 0.05% Tween20 (PBST) and 1% BSA were mixed with 10 nM b-HGFR-Fc for 1 hr. Phagebound to b-HGFR-Fc were captured on neutravidin coated wells for 10 minand unbound phage were washed away with PBST. Phage were eluted using 20mM HCl, 500 mM KCl for 45 m, neutralized, and propagated in XL1 bluecells (Stratagene) in the presence of KO7 helper phage (New EnglandBiolabs). Subsequent rounds of sorting were performed similarly with thefollowing exceptions: in round 2 the final b-HGFR-Fc concentration was5.6 nM, in round 3 the final b-HGFR-Fc concentration was 0.1 nM, inround 4 the final b-HGFR-Fc concentration was 0.5 nM and 780 nMunbiotinylated HGFR-Fc was added to the mixture for 1 h prior to captureon neutravidin.

Phage ELISA—MaxiSorp microtiter plates were coated with human HGFR-Fc at5 μg/ml in PBS over night and then blocked with Casein Blocker. Phagefrom culture supernatants were incubated with serially diluted HGFR-Fcin PBST containing 1% BSA in a tissue culture microtiter plate for 1 hafter which 80 μl of the mixture was transferred to the target coatedwells for 15 min to capture unbound phage. The plate was washed withPBST and HRP conjugated anti-M13 (Amersham Pharmacia Biotech) was added(1:5000 in PBST containing 1% BSA) for 40 min. The plate was washed withPBST and developed by adding Tetramethylbenzidine substrate (Kirkegaardand Perry Laboratories, Gaithersburg, Md.). The absorbance at 405 nm wasplotted as a function of target concentration in solution to determinean IC50. This was used as an affinity estimate for the Fab clonedisplayed on the surface of the phage.

Fab Production and Affinity Determination—To express Fab protein foraffinity measurements, a stop codon was introduced between the heavychain and g3 in the phage display vector. Clones were transformed intoE. coli 34B8 cells and grown in AP5 media at 30 C (Presta et al. CancerRes. 57: 4593-4599 (1997)). Cells were harvested by centrifugation,suspended in 10 mM Tris, 1 mM EDTA pH 8 and broken open using amicrofluidizer. Fab was purified with Protein G affinity chromatography.

Affinity determinations were performed by surface plasmon resonanceusing a BIAcore™-2000. HGFR-Fc was immobilized (1000 response units(RU)) on a CM5 chip and varied concentrations of Fab (4 to 500 nM) inPBST were injected. After each injection the chip was regenerated using100 mM HCl. Binding response was corrected by subtracting the RU from ablank flow cell. A 1:1 Languir model of simultaneous fitting of k_(on)and k_(off) was used for kinetics analysis.

Electro-Chemiluminescent Assay for OA5D5 Blocking of HGF/cMet Binding

Purified cMet-Ig protein produced at Genentech (South San Francisco,Calif.) was biotinylated by incubating with 20-fold molar excessNHS-X-Biotin in 0.1 M NaHCO₃, pH 8.5 using biotin-X-NHS (ResearchOrganics, Cleveland, Ohio). Purified human 2-chain HGF produced atGenentech was labeled with BV-TAG (cat #110034) via NHS-ester chemistryaccording to manufacturer's directions (BioVeris International,Gaithersburg, Md.). cMet-Ig-biotin (500 ng/mL), HGF-Ruthenium Tag (250ng/mL), and titrations of OA5D5 antibody ranging from 3333-0.21 nM ofantibody were incubated together in a volume of 100 ul of assay diluent:PBS+0.5% BSA/0.5% Tween 20/0.033% Proclin (Supelco Inc. Bellefonte Pa.).The mixtures were incubated in sealed polypropylene round bottom 96 wellplates (Corning) for 2-4 hours at room temperature with shakingStreptavidin magnetic beads (Dynabeads, BioVeris) were added. Followinga final 45 min incubation with vigorous shaking, the plates were readusing a BioVeris M-Series instrument (BioVeris International,Gaithersburg, Md.).

KIRA (HGF-Dependent-Met Phosphorylation)

A549 cells (ATCC, Manassas, Va.) were maintained in growth medium (Ham'sF12/DMEM 50:50 [Gibco, Grand Island, N.Y.] containing 10% fetal bovineserum (FBS, Sigma, St. Louis, Mo.). To prepare cells for the assay,cells from confluent cultures were detached using Accutase (ICN, Aurora,Ohio) and seeded into 96 well plates at a density of 50,000 cells perwell. After an overnight incubation at 37° C., growth media was removedand cells were serum starved for 30-60 min in medium containing 0.1%FBS. To determine the ability of OA-5D5 to inhibit cMet phosphorylation,the molecule was serially diluted from 200 to 0.19 nM in medium+0.1% FBSand added to the assay plates. After a 15 min incubation at 37° C., HGF(50 ng/ml) was added. The plates were then incubated for an additional10 minutes at 37° C., the media was removed and a cell lysis buffer wasadded (Cell Signaling Technologies, Cat #9803, Beverly, Mass.;supplemented with a protease inhibitor cocktail purchased fromCalbiochem, Cat #539131, San Diego, Calif.). The lysates were analyzedfor phosphorylated c-Met via an electrochemiluminescence assay using anBioVeris M-Series instrument (BioVeris International, Gaithersburg,Md.). An anti-phosphotyrosine mAb (clone 4G10, Upstate, Lake Placid, MY)was labeled with BV-TAG via NHS-ester chemistry according tomanufacturer's directions (BioVeris). Antibodies against the c-Metextracellular domain were biotinylated using biotin-X-NHS (ResearchOrganics, Cleveland, Ohio). The BV-TAG-labeled 4G10 and biotinylatedanti-c-Met mAb were diluted in assay buffer (PBS/0.5% Tween-20/0.5% BSA)and added as a cocktail to the cell lysates. After a 1.5-2 hr incubationat room temperature with vigorous shaking, streptavidin magnetic beads(Dynabeads, BioVeris) were added. Following a final 45 min incubation,the plates were read on the BioVeris instrument.

Cell Culture and Proliferation Assay

BaF3 is a murine IL-3 dependent lymphoid cell that normally does notexpress cMet and does not respond to HGF. However, in BaF3-hMet derivedby transfection with a normal, full-length cDNA for human c-Met (Schwallet al., J. Cell Biol. (1996), 133:709-718), HGF stimulates proliferationand survival in the absence of IL-3. BaF3-hMet and BaF3-neo cells wereroutinely passaged in RPMI 1640, 5% fetal bovine serum, 4l/L-mercapthoethanol, 100 U/ml penicillin, 100 g/ml streptomycinsulfate, 2 mM L-glutamine, and 5% WEHI-conditioned medium as a source ofIL-3. To measure HGF-dependent proliferation the number of cells after 3days of treatment was quantitated by adding 25 μl Alarma Blue (TrekDiagnostic Systems; Cleveland, Ohio) and measuring fluorescenceintensity 6 hours later. Control experiments were proliferation of thesecells in the absence of HGF. H358-PSF2 and HGF-PSF8 cells were passagedin RPMI 1640, 10% fetal bovine serum, 100 U/ml penicillin, 100 g/mlstreptomycin sulfate, 2 mM L-glutamine. The assay medium was RPMI 1640plus 0.1%, 0.5% BSA, or 10% FBS respectively. The assay was performed asdescribed above.

Immunoprecipitation and Western Blot

H358 cells are a cell line derived from human non-small cell lungcarcinoma (NSCLC). H358-PSF2, H358-PSF8 cells are human HGF stabletransfected H358 cells (Tsao et al., Neoplasia, Vol. 2, No. 3, 2000),and were cultured in RPMI 1640, 10% fetal bovine serum, 100 U/mlpenicillin, 100 g/ml streptomycin sulfate, 2 mM L-glutamine. cMettyrosine phosphorylation detection was performed essentially asdescribed previously (Zioncheck, J Bio Chem, 270(28):16871-8, 1995). Inbrief, cells were plated in 60-mm plates overnight, and medium waschanged to RPMI 1640 containing 0.5% BSA, before adding the combinationsof with or without 1 nM HGF or competitor OA5D5.v2 antibody. After 10min at 37° C., medium was removed and cells were lysed using lysisbuffer (150 mM NaCl, 1.5 mM MgCl₂, 1% Triton X-100, 1× proteaseinhibitor cocktail, 1× phosphatase inhibitor cocktail (Sigma, St. Louis,Mo.)). After spinning, the supernatant of the lysate was incubated withanti-cMet IgG polyclonal antibody (c-28; Santa Cruz Biotechnology, SantaCruz, Calif.) bound to protein G-Sepharose for 1 hour at 4° C. Theimmune complexes were washed and boiled in 1× sample buffer, beforeseparation by SDS-PAGE and electroblotting to nitrocellulose.Phosphotyrosine-containing proteins were visualized using ananti-phosphotyrosine antibody (4G10; Upstate Biotechnology, Waltham,Mass.) followed by HRP-conjugated goat anti-mouse Fab (1:10,000; JacksonLabs, West Grove, Pa.), and in the case of total cMet using c-28antibody (1:400; Santa Cruz Biotechnology, Santa Cruz, Calif.) followedby goat anti-rabbit Fc-HRP (1:10,000; Jackson Labs, West Grove, Pa.)with chemiluminescence detection.

Tumor Xenograft Study

Athymic female mice were inoculated subcutaneously with 5 million KP4pancreatic carcinoma cells. When tumors reached 150-200 mm³, mice wereassigned to 2 groups of 10. Group 1 was injected IP with vehicle twiceper week. Group 2 was injected IP with OA5D5.v2, 30 mg/kg, twice perweek. Tumor size was measured twice per week. Mice were sacrificed whentumor volume exceeded two-times the starting tumor volume, or if thetumor ulcerated.

Results and Discussion

Humanization of 5D5—The human acceptor framework used for thehumanization of 5D5 comprises the consensus human kappa I VL domain anda variant of the human subgroup III consensus VH domain. The variant VHdomain has 3 changes from the human consensus: R71A, N73T and L78A. TheVL and VH domains of murine 5D5 were aligned with the human kappa I andsubgroup III domains; each HVR was identified and then grafted into thehuman acceptor framework to generate a 5D5 graft that could be displayedas an Fab on phage. When phage displaying the 5D5 graft were tested forbinding to immobilized HGFR-Fc, no binding was observed.

A library was generated in which each of the HVR regions of the 5D5graft was soft randomized. This library was panned against immobilizedHGFR-Fc for 4 rounds of selection. Clones were picked for DNA sequenceanalysis and revealed a single clone had been selected. This clone had asingle change in the VH domain at position 94 (R94S) just outside theintended region of HVR-H3 targeted for mutagenesis. Analysis of thisclone by phage ELISA indicated it had similar affinity to that of themonovalent affinity of murine 5D5. When expressed as an Fab and testedby Biacore, the Kd was determined to be 9.8 nM compared to 8.3 nM forthe monovalent affinity of murine 5D5. Thus this unexpected substitutionrestores full binding affinity to the 5D5 graft, and the 5D5 graft plusR94S (hu5D5.v1) represents a fully humanized antibody. Interestingly, ahomologous amino acid, threonine, is found at this position in themurine antibody. MacCallum et al. (MacCallum et al. J. Mol. Biol. 262:732-745 (1996)) have analyzed antibody and antigen complex crystalstructures and found positions 93 and 94 of the heavy chain are part ofthe contact region thus it seems reasonable to include these positionsin the definition of hypervariable region of H3 (HVR-H3) when humanizingantibodies.

Affinity maturation of hu5D5.v1—To improve the affinity of hu5D5.v1, sixphage display libraries were generated in the background of hu5D5.v1,each targeting a single HVR for soft randomization. To avoidre-selecting hu5D5.v1 from a potential high background of wild-typetemplate, stop codons were introduced into the HVR to be mutated priorto generating each library. A solution sorting method was used toenhance the efficiency of the affinity-based phage selection process. Bymanipulating the biotinylated target concentration, reducing the phagecapture time to lower backgrounds and the addition of unbiotinylatedtarget to eliminate clones with faster off rates, high affinity clonescan be proficiently selected. Lee et al., supra. From the first round ofselection, enrichment (target dependent phage capture) was observedsuggesting a large number of clones were present in each library withreasonably high affinity for HGFR-Fc. Selection stringency (see Methodsabove) was increased in subsequent rounds and at round 3 all 6 librarieswere combined to generate a seventh library pool. After 4 rounds ofselection, clones from each of the 7 library pools were analyzed. Allclones in the libraries targeting HVR-L1 and HVR-L3 were identical tohu5D5.v1; however, new sequences were observed in libraries targetingHVR-L2, HVR-H1, HVR-H2 and HVR-H3 (FIG. 2). The library pool consistingof the combination of all 6 libraries was dominated by sequences fromthe HVR-H3 library suggesting that these sequences provided the largestimprovement in affinity for HGFR-Fc (FIG. 3). Selected clones werescreened by phage ELISA and then expressed as Fab protein and theiraffinity determined using Biacore. Several clones from the combinedlibrary with changes in HVR-H3 had improved affinities compared tohu5D5.v1 or the murine 5D5 monovalent affinity (FIG. 4). These cloneshad either S/T at position 94, R/S at position 96 and T/S at position100 and P/S/A at position 100a. The best clone, clone 78 (hu5D5.v2) had3 changes from hu5D5.v1 (94T, 96R and 100T) and a 13-fold affinityimprovement.

Thus starting from the graft of the 6 murine 5D5 HVRs into the humanacceptor scaffold, the expansion of HVR-H3 to include position 94(Threonine) and the addition of 2 changes in HVR-H3 leads to a fullyhuman 5D5 antibody with 13-fold improved binding affinity for HGFR.Furthermore, selected humanized antibodies described herein have beendetermined to have at least comparable biological activity as the parent5D5 antibody, for example in receptor phosphorylation assays, etc. (datanot shown).

Characterization of an antibody of the invention—“One-armed” (alsoreferred to as “one-arm” and “OA”) anti-Met antibodies werecharacterized. Two antibodies of the invention were tested.Specifically, the “OA-5D5.v2” antibody comprised a single Fab armcomprising variable domain sequences as depicted in FIG. 13, wherein theFab arm was fused to an Fc region, and wherein the Fc region was acomplex between one Fc polypeptide comprising the Fc sequence depictedin FIG. 13 and one Fc polypeptide comprising the Fc sequence depicted inFIG. 14. The antibodies were characterized as follows:

-   -   (1) In an assay to test ability of OA-5D5.v2 to block binding of        HGF to its receptor, OA-5D5.v2 was able to block HGF binding to        its receptor at least as well as two comparator        antibodies—namely a chimeric one-armed antibody (which comprised        a Fab arm from the murine parent 5D5 antibody (variable domains        depicted in FIG. 7) fused to a human Fc region), and another        antibody of the invention (OA-5D5.v1). When tested across an        antibody concentration range of about 3333 to 0.21 nM, under        conditions as described in the Materials and Methods section        above, OA-5D5.v2 was found to have an IC50 value that was less        than about half that of a comparator antibody such as the        chimeric one-armed antibody and OA-5D5.v1. Notably, OA-5D5.v1        also blocked with better IC50 than the reference chimeric        antibody. See FIG. 8.    -   (2) In an assay to test ability of OA-5D5.v2 to inhibit HGF        receptor activation, OA-5D5.v2 was able to inhibit kinase        receptor activation at least as well as the two comparator        antibodies as described in (1) above. When tested across an        antibody concentration range of about 200 to 0.19 nM, under        conditions as described in the Materials and Methods section        above, OA-5D5.v2 was found to have an IC50 with a value that was        less than about half that of a comparator antibody such as the        chimeric one-armed antibody and OA-5D5.v1. See FIG. 9.    -   (3) OA-5D5.v2 was also tested for cross-species binding among        human, primate (cynomolgus monkey), canine and murine (mouse).        OA-5D5.v2 was found to bind specifically to human and primte        (cynomolguls monkey) HGF receptor, but not canine or murine        (mouse). (data not shown.)    -   (4) OA-5D5.v2 was tested for its ability to inhibit cell        proliferation in the presence of HGF. As shown in FIG. 10,        OA-5D5.v2 inhibited cell proliferation at least as well as its        chimeric antibody counterpart and OA-5D5.v1 (as described in (1)        above). When tested across an antibody concentration range of        about 0.01 to 100 nM, under conditions as described in the        Materials and Methods section above, OA-5D5.v2 was found to have        an IC50 value that was less than about half that of a comparator        antibody such as the chimeric one-armed antibody and OA-5D5.v1.        See FIG. 10. Specific binding of OA-5D5.v2 to the        Met-transfected cells was confirmed by FACs analysis. (data not        shown)    -   (5) OA-5D5.v2 was tested for its ability to inhibit receptor        tyrosine phosphorylation in the presence of HGF. As shown in        FIGS. 11A and B, OA-5D5.v2 inhibited receptor tyrosine        phosphorylation when tested at antibody concentrations from        about 10 to 1000 nM. See FIGS. 11A and B.    -   (6) OA-5D5.v2 was tested for in vivo efficacy using a tumor        xenograft model based on a pancreatic tumor cell line (KP4).        Results from this efficacy study showed that the OA-5D5.v2        antibody was capable of inhibiting and causing regression of        tumors in vivo. As shown in FIG. 12, there was complete loss of        tumor in most of the animals treated with the antibody.

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1. An anti-c-met antibody comprising: (a) at least one HVR sequenceselected from the group consisting of: (i) HVR-L1 comprising sequenceA1-A17, wherein A1-A17 is KSSQSLLYTSSQKNYLA (SEQ ID NO:1) (ii) HVR-L2comprising sequence B1-B7, wherein B1-B7 is WASTRES (SEQ ID NO:2) (iii)HVR-L3 comprising sequence C1-C9, wherein C1-C9 is QQYYAYPWT (SEQ IDNO:3) (iv) HVR-H1 comprising sequence D1-D10, wherein D1-D10 isGYTFTSYWLH (SEQ ID NO:4) (v) HVR-H2 comprising sequence E1-E18, whereinE1-E18 is GMIDPSNSDTRFNPNFKD (SEQ ID NO:5) (vi) HVR-H3 comprisingsequence F1-F 11, wherein F1-F11 is XYGSYVSPLDY (SEQ ID NO:6) and X isnot R; and (b) at least one variant HVR, wherein the variant HVRcomprises modification of at least one residue of the sequence depictedin SEQ ID NO: 1, 2, 3, 4, 5 or
 6. 2. The antibody of claim 1, wherein Flin a variant HVR-H3 is T or S, F3 is R or S and F7 is T.
 3. The antibodyof claim 1, wherein the antibody is humanized.
 4. The antibody of claim1, wherein at least a portion of the framework sequence is a humanconsensus framework sequence.
 5. The antibody of claim 1, wherein saidmodification is substitution, insertion or deletion.
 6. The antibody ofclaim 1, wherein a HVR-L2 variant comprises 1-5 (1, 2, 3, 4 or 5)substitutions in any combination of the following positions: B1 (M orL), B2 (P, T, G or S), B3 (N, G, R or T), B4 (I, N or F), B5 (P, I, L orG), B6 (A, D, T or V) and B7 (R, I, M or G).
 7. The antibody of claim 1,wherein a HVR-H1 variant comprises 1-5 (1, 2, 3, 4 or 5) substitutionsin any combination of the following positions: D3 (N, P, L, S, A, I), D5(I, S or Y), D6 (G, D, T, K, R), D7 (F, H, R, S, T or V) and D9 (M orV).
 8. The antibody of claim 1, wherein a HVR-H2 variant comprises 1-4(1, 2, 3 or 4) substitutions in any combination of the followingpositions: E7 (Y), E9 (I), E10 (I), E14 (T or Q), E15 (D, K, S, T or V),E16 (L), E17 (E, H, N or D) and E18 (Y, E or H).
 9. The antibody ofclaim 1, wherein a HVR-H3 variant comprises 1-5 (1, 2, 3, 4 or 5)substitutions in any combination of the following positions: F1 (T, S),F3 (R, S, H, T, A, K), F4 (G), F6 (R, F, M, T, E, K, A, L, W), F7 (L, I,T, R, K, V), F8 (S, A), F10 (Y, N) and F11 (Q, S, H, F).
 10. Theantibody of claim 1 comprising an HVR-L1 having the sequence of SEQ IDNO:1.
 11. The antibody of claim 1 comprising an HVR-L3 having thesequence of SEQ ID NO:3.
 12. The antibody of claim 1, wherein F1 in avariant HVR-H3 is T.
 13. The antibody of claim 1, wherein F3 in avariant HVR-H3 is R or S.
 14. The antibody of claim 1, wherein F7 in avariant HVR-H3 is T.
 15. A humanized anti-c-met antibody whereinmonovalent affinity of the antibody to human c-met is substantially thesame as monovalent affinity of a murine antibody comprising a lightchain and heavy chain variable sequence as depicted in FIG. 7 (SEQ IDNO: 9 and 10).
 16. A humanized anti-c-met antibody wherein monovalentaffinity of the antibody to human c-met is at least 3-fold greater thanmonovalent affinity of a murine antibody comprising a light chain andheavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO: 9 and10).
 17. The humanized antibody of claim 15 or 16 wherein the murineantibody is produced by hybridoma cell line deposited under AmericanType Culture Collection Accession Number ATCC with designation HB-11894(hybridoma 1A3.3.13) or HB-11895 (hybridoma 5D5.11.6).
 18. The antibodyof any of claims 15-17 wherein the binding affinity is expressed as a Kdvalue.
 19. The antibody of any of claim 15-18 wherein the bindingaffinity is measured by Biacore or radioimmunoassay.
 20. The antibody ofclaim 1 comprising human subgroup 1 consensus framework sequence. 21.The antibody of claim 1 comprising heavy chain human subgroup IIIconsensus framework sequence.
 22. The antibody of claim 21 wherein theframework sequence comprises a substitution at position 71, 73 and/or78.
 23. The antibody of claim 22 wherein said substitution is R71A, N73Tand/or N78A.
 24. A humanized anti-c-met antibody wherein the humanizedantibody inhibits binding human hepatocyte growth factor to its receptorbetter than a reference antibody comprising a chimeric anti-c-metantibody comprising a light chain and heavy chain variable sequence asdepicted in FIG. 7 (SEQ ID NO: 9 and 10).
 25. The antibody of claim 24,wherein the humanized antibody inhibits binding with an IC50 value thatis less than half that of the chimeric antibody.
 26. The antibody ofclaim 25, wherein the IC50 is determined across an antibodyconcentration range from about 0.01 nM to around 1000 nM.
 27. Ahumanized anti-c-met antibody wherein the humanized antibody inhibitshuman hepatocyte growth factor (HGF) receptor activation better than areference antibody comprising a chimeric anti-c-met antibody comprisinga light chain and heavy chain variable sequence as depicted in FIG. 7(SEQ ID NO: 9 and 10).
 28. The antibody of claim 27, wherein thehumanized antibody inhibits receptor activation with an IC50 value thatis less than half that of the chimeric antibody.
 29. The antibody ofclaim 28, wherein the IC50 is determined across an antibodyconcentration range from about 0.1 nM to about 100 nM.
 30. A humanizedanti-c-met antibody wherein the humanized antibody inhibitsc-met-dependent cell proliferation better than a reference antibodycomprising a chimeric anti-c-met antibody comprising a light chain andheavy chain variable sequence as depicted in FIG. 7 (SEQ ID NO: 9 and10).
 31. The antibody of claim 30, wherein the humanized antibodyinhibits cell proliferation with an IC50 value that is less than halfthat of the chimeric antibody.
 32. The antibody of claim 31, wherein theIC50 is determined across an antibody concentration range from about0.01 nM to about 100 nM.
 33. The antibody of the preceding claims,wherein both the humanized antibody and chimeric antibody aremonovalent.
 34. The antibody of the preceding claims, wherein both thehumanized antibody and chimeric antibody comprise a single Fab regionlinked to an Fc region.
 35. An antibody comprising a heavy chainvariable domain comprising HVR1-HC, HVR2-HC and/or HVR3-HC sequencedepicted in FIG. 13 (SEQ ID NO: 191-193).
 36. The antibody of claim 35,wherein the variable domain comprises FR1-HC, FR2-HC, FR3-HC and/orFR4-HC sequence depicted in FIG. 13 (SEQ ID NO: 187-190).
 37. Theantibody of claim 35 or 36, wherein the antibody comprises CH1 and/or Fcsequence depicted in FIG. 13 (SEQ ID NO: 194 and/or 195).
 38. Anantibody comprising a light chain variable domain comprising HVR1-LC,HVR2-LC and/or HVR3-LC sequence depicted in FIG. 13 (SEQ ID NO:183-185).
 39. The antibody of claim 38, wherein the variable domaincomprises FR1-LC, FR2-LC, FR3-LC and/or FR4-LC sequence depicted in FIG.13 (SEQ ID NO: 179-182).
 40. The antibody of claim 38 or 39, wherein theantibody comprises CL1 sequence depicted in FIG. 13 (SEQ ID NO: 186).41. An antibody comprising a heavy chain variable domain of any ofclaims 35-37 and a light chain variable domain of any of claims 38-40.42. The antibody of claim 41, wherein the antibody is monovalent andcomprises an Fc region.
 43. The antibody of claim 42, wherein the Fcregion comprises a first and a second polypeptide, wherein the first andsecond polypeptide each comprises one or more mutations with respect towild type human Fc.
 44. The antibody of claim 43, wherein the firstpolypeptide comprises the Fc sequence depicted in FIG. 13 (SEQ ID NO:195) and the second polypeptide comprises the sequence depicted in FIG.14 (SEQ ID NO: 196).
 45. A method of inhibiting c-met activated cellproliferation, said method comprising contacting a cell or tissue withan effective amount of an antibody of any of the preceding claims.
 46. Amethod of modulating a disease associated with dysregulation of theHGF/c-met signaling axis, said method comprising administering to asubject an effective amount of an antibody of any of the precedingclaims.
 47. A method of treating a subject having cancer, said methodcomprising administering to the subject an effective amount of anantibody of any of the preceding claims.
 48. The method of claim 47,wherein the cancer is lung cancer, brain cancer, kidney cancer, gastriccancer, colorectal cancer and/or pancreatic cancer.
 49. A method oftreating a proliferative disorder in a subject, said method comprisingadministering to the subject an effective amount of an antibody of anyof the preceding claims.
 50. The method of claim 49, wherein theproliferative disorder is cancer.
 51. A nucleic acid encoding theantibody of any of claims 1-44.
 52. A host cell comprising the nucleicacid of claim
 51. 53. A composition comprising the antibody of any ofclaims 1-44.
 54. The composition of claim 53, wherein the compositioncomprises a carrier.